Use of CD2/5/7 knock-out anti-CD2/5/7 chimeric antigen receptor T cells against T cell lymphomas and leukemias

ABSTRACT

The present invention includes compositions and methods for treating T cell lymphomas and leukemias. In certain aspects, the compositions and methods include CAR T cells targeting CD2, CD5, or CD7 and modified cells wherein CD2, CD5, or CD7 has been knocked-out.

CROSS REFERENCE TO RELATED APPLICATION

The present application is a 35 U.S.C. § 371 national phase applicationfrom, and claims priority to, International Application No.PCT/US2019/067613, filed Dec. 19, 2019, and published under PCT Article21(2) in English, which claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 62/782,131, filed Dec. 19, 2018,which are hereby incorporated by reference in their entireties herein.

BACKGROUND OF THE INVENTION

T cell lymphomas and leukemias are aggressive neoplasms derived from Tcell progenitors or differentiated T cells. Mature or peripheral T celllymphomas account for 10%-15% of all non-Hodgkin's lymphomas, or˜7,000-10,000 cases in the U.S. per year. T cell lymphomas and leukemiashave poor prognoses and there are few available treatments. Chimericantigen receptor T cell (CART) therapy has demonstrated efficacy for Bcell neoplasms, but extending the success of CAR T cells to T cellmalignancies is problematic because most target antigens are sharedbetween normal and malignant cells, leading to CAR T cell fratricide.

A need exists for compositions and methods for treating T cell lymphomasand leukemias as well as methods for eliminating CAR T cell fratricide.The present invention addresses this need.

SUMMARY OF THE INVENTION

As described herein, the present invention relates to compositions andmethods utilizing CAR T cells targeting CD2, CD5, or CD7 and modifiedcells wherein CD2, CD5, or CD7 has been knocked-out.

In one aspect, the invention includes a method of treating cancer in asubject in need thereof. The method comprises administering to thesubject a first modified cell comprising a chimeric antigen receptor(CAR), wherein the CAR comprises an antigen binding domain, atransmembrane domain, and an intracellular domain, and administering tothe subject a second modified cell wherein the endogenous CD5 gene hasbeen knocked-out.

In another aspect, the invention includes a method of treating cancer ina subject in need thereof. The method comprises administering to thesubject a first modified cell comprising a chimeric antigen receptor(CAR), wherein the CAR comprises an antigen binding domain capable ofbinding CD2, a transmembrane domain, and an intracellular domain, andadministering to the subject a second modified cell wherein theendogenous CD2 gene has been knocked-out.

In yet another aspect, the invention includes a method of treatingcancer in a subject in need thereof. The method comprises administeringto the subject a first modified cell comprising a CAR, wherein the CARcomprises an antigen binding domain capable of binding CD5, atransmembrane domain, and an intracellular domain, and administering tothe subject a second modified cell wherein the endogenous CD5 gene hasbeen knocked-out.

In still another aspect, the invention includes a method of treatingcancer in a subject in need thereof comprising administering to thesubject a first modified cell comprising a CAR, wherein the CARcomprises an antigen binding domain capable of binding CD7, atransmembrane domain, and an intracellular domain, and administering tothe subject a second modified cell wherein the endogenous CD7 gene hasbeen knocked-out.

Another aspect of the invention includes a nucleic acid comprising aCAR, wherein the CAR comprises an antigen binding domain capable ofbinding CD2, a transmembrane domain, and an intracellular domain.

Yet another aspect of the invention includes a nucleic acid comprising aCAR, wherein the CAR comprises an antigen binding domain capable ofbinding CD5, a transmembrane domain, and an intracellular domain.

Still another aspect of the invention includes a vector comprising anyof the nucleic acids disclosed herein.

In another aspect, the invention includes a cell comprising any of thenucleic acids disclosed herein or any of the vectors disclosed herein.

In yet another aspect, the invention includes a composition comprising afirst modified cell comprising a chimeric antigen receptor (CAR),wherein the CAR comprises an antigen binding domain that targets CD2, atransmembrane domain, and an intracellular domain, and a second modifiedcell wherein the endogenous CD2 gene has been knocked-out.

In still another aspect, the invention includes a composition comprisinga first modified cell comprising a chimeric antigen receptor (CAR),wherein the CAR comprises an antigen binding domain that targets CD5, atransmembrane domain, and an intracellular domain, and a second modifiedcell wherein the endogenous CD5 gene has been knocked-out.

In another aspect, the invention includes a composition comprising afirst modified cell comprising a chimeric antigen receptor (CAR),wherein the CAR comprises an antigen binding domain that targets CD7, atransmembrane domain, and an intracellular domain, and a second modifiedcell wherein the endogenous CD7 gene has been knocked-out.

Another aspect of the invention includes a composition comprising afirst modified cell comprising a chimeric antigen receptor (CAR),wherein the CAR comprises an antigen binding domain, a transmembranedomain, and an intracellular domain, and a second modified cell whereinthe endogenous CD5 gene has been knocked-out.

In various embodiments of the above aspects or any other aspect of theinvention delineated herein, the endogenous gene is knocked-out using aCRISPR method. In certain embodiments, the CRISPR method is aCRISPR/Cas9 method. In certain embodiments, the CRISPR/Cas9 methodutilizes an sgRNA comprising the nucleotide sequence of SEQ ID NO: 23.In certain embodiments, the CRISPR/Cas9 method utilizes an sgRNAcomprising the nucleotide sequence selected from the group consisting ofSEQ ID NO: 22-24. In certain embodiments, the antigen binding domain ofthe CAR is capable of binding an antigen selected from the groupconsisting of CD5, CD19, CD2, CD7, a tumor-specific antigen (TSA), atumor associated antigen (TAA), a glioma-associated antigen,carcinoembryonic antigen (CEA), β-human chorionic gonadotropin,alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1,MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS),intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase,prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein,PSMA, Her2/neu, survivin, telomerase, prostate-carcinoma tumor antigen-1(PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulingrowth factor (IGF)-I, IGF-II, IGF-I receptor, mesothelin, MART-1/MelanA(MART-I), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3,BAGE, GAGE-1, GAGE-2, p15, Ras, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK,MYL-RAR, EBVA, HPV antigen E6, HPV antigen E7, TSP-180, MAGE-4, MAGE-5,MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA,TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4,Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG,BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50,CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50,MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 bindingprotein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.

In certain embodiments, the antigen binding domain of the CAR comprisesa complementarity determining region (CDR) comprising the amino acidsequence selected from the group consisting of SEQ ID NOs: 31-36, 43-48,53-58, 65-70, 83-88, and 95-100. In certain embodiments, the antigenbinding domain of the CAR comprises a complementarity determining region(CDR) comprising the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 31-36, 43-48, 53-58, and 65-70. In certainembodiments, the antigen binding domain of the CAR comprises acomplementarity determining region (CDR) comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 83-88 and95-100.

In certain embodiments, the antigen binding domain of the CAR comprisesa heavy chain variable region comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 29, 41, 51, 63, 75, 81, and 93.In certain embodiments, the antigen binding domain of the CAR comprisesa light chain variable region comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 30, 42, 52, 64, 76, 82, and 94.

In certain embodiments, the antigen binding domain of the CAR comprisesa heavy chain variable region comprising the amino acid sequenceselected from the group consisting of SEQ ID NOs: 29, 41, 51, and 63. Incertain embodiments, the antigen binding domain of the CAR comprises alight chain variable region comprising the amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 30, 42, 52, and 64.

In certain embodiments, the antigen binding domain of the CAR comprisesa heavy chain variable region comprising the amino acid sequenceselected from the group consisting of SEQ ID NOs: 75, 81, and 93. Incertain embodiments, the antigen binding domain of the CAR comprises alight chain variable region comprising the amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 76, 82, and 94.

In certain embodiments, the antigen binding domain of the CAR comprisesan scFv comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 27, 28, 39, 40, 50, 61, 62, 73, 74, 79, 80,91, and 92. In certain embodiments, the antigen binding domain of theCAR comprises an scFv comprising the amino acid sequence selected fromthe group consisting of SEQ ID NOs: 27, 28, 39, 40, 50, 61, and 62. Incertain embodiments, the antigen binding domain of the CAR comprises anscFv comprising the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 73, 74, 79, 80, 91, and 92.

In certain embodiments, the CAR comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 25, 26, 37, 38, 49,59, 60, 71, 72, 77, 78, 89, and 90. In certain embodiments, the CARcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 25, 26, 37, 38, 49, 59, and 60. In certain embodiments, theCAR comprises an amino acid sequence selected from the group consistingof SEQ ID NOs: 71, 72, 77, 78, 89, and 90.

In certain embodiments, the CAR is encoded by a nucleic acid sequenceselected from the group consisting of SEQ ID NOs: 1-13. In certainembodiments, the CAR is encoded by a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs: 1-7. In certain embodiments, the CARis encoded by a nucleic acid sequence selected from the group consistingof SEQ ID NOs: 8-13.

In certain embodiments, the CAR further comprises a suicide gene. Incertain embodiments, the suicide gene is iCaspase9.

In certain embodiments, the first and or second modified cell is a Tcell.

In certain embodiments, the cancer comprises a T cell lymphoma or a Tcell leukemia. In certain embodiments, the cancer is selected from thegroup consisting of acute myeloid leukemia (AML), T-cell acutelymphoblastic leukemia (T-ALL), acute lymphoblastic leukemia (ALL), andchronic lymphocytic leukemia (CLL).

In certain embodiments, the composition comprises a pharmaceuticallyacceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of specific embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings exemplary embodiments. It should beunderstood, however, that the invention is not limited to the precisearrangements and instrumentalities of the embodiments shown in thedrawings.

FIG. 1 is a schematic illustrating current issues with CART therapy forT cell neoplasms.

FIG. 2 is a schematic illustrating development of an innovative strategywhere: i. a tumor target is removed from normal T cells usinggene-editing and thereby avoids fratricide during manufacturing; ii. asecond T cell product containing normal T cells wherein the T celltarget is knocked-out (KO) that is co-infused with an anti-T cellneoplasm CART to provide T cell immunity that is not affected by CARTkilling.

FIG. 3 is a schematic illustrating a novel approach to target T celllymphomas without causing T cell toxicity. A two-pronged immunotherapyis used that includes anti-T-NHL (or T-ALL) CART (either CART2/5/7, e.g.CART2) and CD2/5/7 knocked out normal T cells. The CART will destroytumor cells but will also kill normal T cells. The infusion of CD2/5/7(or other tumor target) KO normal T cells will provide CART-resistant Tcell immunity until the CART cells are depleted.

FIG. 4 illustrates anti-CD2 and anti-CD5 CAR constructs used herein. Allconstructs have a lentiviral pTRPE 4-1BB CD3zeta backbone.

FIG. 5 illustrates CART transduction efficiency in T cells. Sixdifferent CAR5 constructs were generated using single-chain variablefragments (scFvs) with high (#17), medium (#34) and low (#9) affinity. Tcells were activated with anti-CD3/CD28 beads (Dynabeads) and 24 hourslater lentiviral vectors were added at a MOI of 3. Dynabeads wereremoved at day 6. CART cells were frozen when the mean volume was below350 fl. CAR expression (goat-anti-mouse Fab antibody) was tested at day6.

FIG. 6 illustrates the CD5 (or CD2, or CD7) KO manufacturing process andCRISPR-Cas9 KO efficiency.

FIG. 7 illustrates expansion curves of several CART groups. Without CD2KO CART2 cells would not expand. With KO CART2 and CART5 reach about 5-8population doublings.

FIG. 8 illustrates the finding that without CRISPR-Cas9 KO of CD5, theCD5 mean fluorescence intensity (MFI) was 10 folds less in CART5 ascompared to control T cells, while there was no change in another panT-cell marker such as CD2.

FIG. 9 illustrates CART2 and CART5 expansion curves. T cellconcentration was measured using Coulter Counter.

FIG. 10 illustrates results from an experiment wherein six differentCAR2 constructs were challenged in vitro by co-culturing them withluciferase+ Jurkat cells (T-cell leukemia cell line). At 24 hours, totalkilling was measured as relative reduction in luminescence. Only C3029,C3030 and C3043 showed anti-tumor effects.

FIG. 11 illustrates results from an experiment wherein six differentCAR5 constructs were challenged in vitro by co-culturing them withluciferase+ Jurkat cells (T-cell leukemia cell line). At 24 hours, totalkilling was measured as relative reduction in luminescence. All the CAR5constructs showed similar anti-tumor effects.

FIG. 12 illustrates the in vivo efficacy of CART2 and CART5. NSG micewere engrafted with Luciferase+ Jurkat cells and mice were randomized toreceive control T cells or CART2 or CART5 (1×10⁶) at day 7. Mice wereimaged weekly using the IVIS Xenogen Spectrum and analyzed withLivingImage software. CART2 C3043 and CART5 C3054 were the mosteffective.

FIG. 13 illustrates results from an experiment wherein Jurkat cells weretransduced with different CAR5 constructs (targeted epitope and affinityshown to the left) and with a GFP-NFAT reporter then co-cultured withCD5+ tumor cells (or controls) for 24 hours. The lead CART5 (C3054)showed increased NFAT activation.

FIG. 14 illustrates results from an experiment wherein Jurkat cells weretransduced with the different CAR2 constructs and with a GFP-NFATreporter then co-cultured with CD2+ tumor cells (or controls) for 24hours. The lead CART2 (C3043) showed increased NFAT activation.

FIG. 15 illustrates CART2 and CART5 activity against cutaneous T celllymphoma. Results from 24-hour killing assays are shown. CART2 cells areactive against primary Sezary cells (leukemic Cutaneous T Cell Lymphoma)and the HH Sezary cell line. Also CART5 were active against HH cells.

FIG. 16 illustrates the finding that CART2 and CART5 can recognizenormal T cells (autologous top and allogeneic bottom) and kill them.

FIG. 17 illustrates the finding that removal of the CAR target protectsnormal T cells from CART killing. CD5 KO but not WT normal T cells areresistant to CART5 killing. Normal resting T cells are recognized andkilled by CART2 (top) and CART5 (bottom). Efficient KO of CD2 or CD5from normal T cells using CRISPR-Cas9 lead to resistance to CART2 orCART5 killing respectively.

FIG. 18 illustrates the finding that CMV-specific T cells are present inCD2KO and CD5KO normal T cell products. CD2 and CD5 KO normal T cellsmaintain the ability to recognize CMV peptides and produce cytokines.(HLA-A-02:01-CMV PP65 NLVPMVATV dextramer (SEQ ID NO: 101); ICS after 4h exposure to CETF peptides. After secondary culture with CMV-peptidepulsed APC).

FIG. 19 illustrates the development of dual specific CART cells. Twolentiviral constructs were generated that included the CAR5 (C3054) andCAR2 (C3043) linked by a P2A sequence. Gene expression is driven by anEFlalpha promoter. The CAR5 constructs have 4-1BB costimulatory andCD3zeta signaling domains.

FIGS. 20A-20B illustrate dual KO CART cells. Efficient knock-out of bothCD2 and CD5 in normal T cells as shown by flow cytometry.

FIG. 21 illustrates the finding that CD5 KO CART5 are more effectivethan CD5+ CART5 in vivo. CD5 KO increases CART5 anti-tumor efficacy. Ina Jurkat T-ALL xenograft model using NSG mice, CD5 KO CART5 (2×10⁶cells/mouse) lead to complete long-term complete responses and longersurvival as compared to WT CART5.

FIG. 22 illustrates the finding that CD5 KO CART19 are more effectivethan CD5+ CART19 in vivo. CD5 KO increases CART19 anti-tumor efficacy.In a NALM6 B-ALL xenograft model, CD5 KO CART19 have drastically highertumor control as compared to WT CART19.

FIGS. 23A-23B illustrate the finding that CART5 and CART2 can target 20%of AML. FIG. 23A illustrates CD2 expression in AML. FIG. 23B illustratesresults from a 24 hour killing assay. CART2 cells were co-cultured withCD2+ AML cells and showed significant killing at 24 hours.

FIG. 24 illustrates the finding that CART5 can target 100% of CLL andMCL. Results are from cytotoxicity assays showing that CART5 cells canrecognize and kill CD5+ MCL cell lines (Jeko-1 and Mino).

FIG. 25 . illustrates development of a sgRNA to knock-out CD7 in T cells(top) and the generation of six CAR constructs against CD7.

FIG. 26 illustrates Casp9-CAR5 lentiviral constructs used herein. Twolentiviral constructs were generated that included the CAR5 (C3054), aP2A sequence and then the iCaspase9 suicide gene (iC9) or iC9-P2A-C3054.Gene expression is driven by an EF1alpha promoter. The CAR5 constructhas 4-1BB costimulatory and CD3zeta signaling domains.

DETAILED DESCRIPTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

“Activation,” as used herein, refers to the state of a T cell that hasbeen sufficiently stimulated to induce detectable cellularproliferation. Activation can also be associated with induced cytokineproduction, and detectable effector functions. The term “activated Tcells” refers to, among other things, T cells that are undergoing celldivision.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which specifically binds with an antigen. Antibodies can beintact immunoglobulins derived from natural sources or from recombinantsources and can be immunoreactive portions of intact immunoglobulins.Antibodies are typically tetramers of immunoglobulin molecules. Theantibodies in the present invention may exist in a variety of formsincluding, for example, polyclonal antibodies, monoclonal antibodies,Fv, Fab and F(ab)₂, as well as single chain antibodies (scFv) andhumanized antibodies (Harlow et al., 1999, In: Using Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow etal., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor,N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883;Bird et al., 1988, Science 242:423-426).

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limitedto, Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, scFvantibodies, and multispecific antibodies formed from antibody fragments.

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations.

An “antibody light chain,” as used herein, refers to the smaller of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations. Kappa and lambda light chainsrefer to the two major antibody light chain isotypes.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to elicit thedesired immune response. Moreover, a skilled artisan will understandthat an antigen need not be encoded by a “gene” at all. It is readilyapparent that an antigen can be generated synthesized or can be derivedfrom a biological sample. Such a biological sample can include, but isnot limited to a tissue sample, a tumor sample, a cell or a biologicalfluid.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

“Allogeneic” refers to any material derived from a different animal ofthe same species.

“Xenogeneic” refers to any material derived from an animal of adifferent species.

The term “chimeric antigen receptor” or “CAR,” as used herein, refers toan artificial T cell receptor that is engineered to be expressed on animmune effector cell and specifically bind an antigen. CAR5 may be usedas a therapy with adoptive cell transfer. T cells are removed from apatient and modified so that they express the receptors specific to aparticular form of antigen. In some embodiments, the CAR5 hasspecificity to a selected target, for example a B cell surface receptor.CAR5 may also comprise an intracellular activation domain, atransmembrane domain and an extracellular domain comprising a tumorassociated antigen binding region. In some aspects, CAR5 comprise anextracellular domain comprising an anti-B cell binding domain fused toCD3-zeta transmembrane and intracellular domain

The term “cleavage” refers to the breakage of covalent bonds, such as inthe backbone of a nucleic acid molecule or the hydrolysis of peptidebonds. Cleavage can be initiated by a variety of methods, including, butnot limited to, enzymatic or chemical hydrolysis of a phosphodiesterbond. Both single-stranded cleavage and double-stranded cleavage arepossible. Double-stranded cleavage can occur as a result of two distinctsingle-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered ends. In certainembodiments, fusion polypeptides may be used for targeting cleaveddouble-stranded DNA.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not significantlyaffect or alter the binding characteristics of the antibody containingthe amino acid sequence. Such conservative modifications include aminoacid substitutions, additions and deletions. Modifications can beintroduced into an antibody of the invention by standard techniquesknown in the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions are ones in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, oneor more amino acid residues within the CDR regions of an antibody can bereplaced with other amino acid residues from the same side chain familyand the altered antibody can be tested for the ability to bind antigensusing the functional assays described herein.

“Co-stimulatory ligand,” as the term is used herein, includes a moleculeon an antigen presenting cell (e.g., an aAPC, dendritic cell, B cell,and the like) that specifically binds a cognate co-stimulatory moleculeon a T cell, thereby providing a signal which, in addition to theprimary signal provided by, for instance, binding of a TCR/CD3 complexwith an MHC molecule loaded with peptide, mediates a T cell response,including, but not limited to, proliferation, activation,differentiation, and the like. A co-stimulatory ligand can include, butis not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL,OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesionmolecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM,lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist orantibody that binds Toll ligand receptor and a ligand that specificallybinds with B7-H3. A co-stimulatory ligand also encompasses, inter alia,an antibody that specifically binds with a co-stimulatory moleculepresent on a T cell, such as, but not limited to, CD27, CD28, 4-1BB,OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specificallybinds with CD83.

A “co-stimulatory molecule” refers to the cognate binding partner on a Tcell that specifically binds with a co-stimulatory ligand, therebymediating a co-stimulatory response by the T cell, such as, but notlimited to, proliferation. Co-stimulatory molecules include, but are notlimited to an MHC class I molecule, BTLA and a Toll ligand receptor.

A “co-stimulatory signal”, as used herein, refers to a signal, which incombination with a primary signal, such as TCR/CD3 ligation, leads to Tcell proliferation and/or upregulation or downregulation of keymolecules.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate. In contrast, a “disorder”in an animal is a state of health in which the animal is able tomaintain homeostasis, but in which the animal's state of health is lessfavorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

The term “downregulation” as used herein refers to the decrease orelimination of gene expression of one or more genes.

“Effective amount” or “therapeutically effective amount” are usedinterchangeably herein, and refer to an amount of a compound,formulation, material, or composition, as described herein effective toachieve a particular biological result or provides a therapeutic orprophylactic benefit. Such results may include, but are not limited to,anti-tumor activity as determined by any means suitable in the art.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The term “expand” as used herein refers to increasing in number, as inan increase in the number of T cells. In one embodiment, the T cellsthat are expanded ex vivo increase in number relative to the numberoriginally present in the culture. In another embodiment, the T cellsthat are expanded ex vivo increase in number relative to other celltypes in the culture. The term “ex vivo,” as used herein, refers tocells that have been removed from a living organism, (e.g., a human) andpropagated outside the organism (e.g., in a culture dish, test tube, orbioreactor).

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., Sendai viruses, lentiviruses, retroviruses,adenoviruses, and adeno-associated viruses) that incorporate therecombinant polynucleotide.

“Homologous” as used herein, refers to the subunit sequence identitybetween two polymeric molecules, e.g., between two nucleic acidmolecules, such as, two DNA molecules or two RNA molecules, or betweentwo polypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit; e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous at that position. The homology between two sequences is adirect function of the number of matching or homologous positions; e.g.,if half (e.g., five positions in a polymer ten subunits in length) ofthe positions in two sequences are homologous, the two sequences are 50%homologous; if 90% of the positions (e.g., 9 of 10), are matched orhomologous, the two sequences are 90% homologous.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a complementary-determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, humanized antibodiescan comprise residues which are found neither in the recipient antibodynor in the imported CDR or framework sequences. These modifications aremade to further refine and optimize antibody performance. In general,the humanized antibody will comprise substantially all of at least one,and typically two, variable domains, in which all or substantially allof the CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin sequence. The humanized antibody optimally also willcomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. For further details, see Joneset al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

“Fully human” refers to an immunoglobulin, such as an antibody, wherethe whole molecule is of human origin or consists of an amino acidsequence identical to a human form of the antibody.

“Identity” as used herein refers to the subunit sequence identitybetween two polymeric molecules particularly between two amino acidmolecules, such as, between two polypeptide molecules. When two aminoacid sequences have the same residues at the same positions; e.g., if aposition in each of two polypeptide molecules is occupied by anArginine, then they are identical at that position. The identity orextent to which two amino acid sequences have the same residues at thesame positions in an alignment is often expressed as a percentage. Theidentity between two amino acid sequences is a direct function of thenumber of matching or identical positions; e.g., if half (e.g., fivepositions in a polymer ten amino acids in length) of the positions intwo sequences are identical, the two sequences are 50% identical; if 90%of the positions (e.g., 9 of 10), are matched or identical, the twoamino acids sequences are 90% identical.

The term “immunoglobulin” or “Ig,” as used herein is defined as a classof proteins, which function as antibodies. Antibodies expressed by Bcells are sometimes referred to as the BCR (B cell receptor) or antigenreceptor. The five members included in this class of proteins are IgA,IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present inbody secretions, such as saliva, tears, breast milk, gastrointestinalsecretions and mucus secretions of the respiratory and genitourinarytracts. IgG is the most common circulating antibody. IgM is the mainimmunoglobulin produced in the primary immune response in most subjects.It is the most efficient immunoglobulin in agglutination, complementfixation, and other antibody responses, and is important in defenseagainst bacteria and viruses. IgD is the immunoglobulin that has noknown antibody function, but may serve as an antigen receptor. IgE isthe immunoglobulin that mediates immediate hypersensitivity by causingrelease of mediators from mast cells and basophils upon exposure toallergen.

The term “immune response” as used herein is defined as a cellularresponse to an antigen that occurs when lymphocytes identify antigenicmolecules as foreign and induce the formation of antibodies and/oractivate lymphocytes to remove the antigen.

When “an immunologically effective amount,” or “therapeutic amount” isindicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician orresearcher with consideration of individual differences in age, weight,tumor size, extent of infection or metastasis, and condition of thepatient (subject).

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the compositions and methods ofthe invention. The instructional material of the kit of the inventionmay, for example, be affixed to a container which contains the nucleicacid, peptide, and/or composition of the invention or be shippedtogether with a container which contains the nucleic acid, peptide,and/or composition. Alternatively, the instructional material may beshipped separately from the container with the intention that theinstructional material and the compound be used cooperatively by therecipient.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

The term “knockdown” as used herein refers to a decrease in geneexpression of one or more genes.

The term “knockout” as used herein refers to the ablation of geneexpression of one or more genes.

A “lentivirus” as used herein refers to a genus of the Retroviridaefamily. Lentiviruses are unique among the retroviruses in being able toinfect non-dividing cells; they can deliver a significant amount ofgenetic information into the DNA of the host cell, so they are one ofthe most efficient methods of a gene delivery vector. HIV, SIV, and FIVare all examples of lentiviruses. Vectors derived from lentivirusesoffer the means to achieve significant levels of gene transfer in vivo.

The term “limited toxicity” as used herein, refers to the peptides,polynucleotides, cells and/or antibodies of the invention manifesting alack of substantially negative biological effects, anti-tumor effects,or substantially negative physiological symptoms toward a healthy cell,non-tumor cell, non-diseased cell, non-target cell or population of suchcells either in vitro or in vivo.

By the term “modified” as used herein, is meant a changed state orstructure of a molecule or cell of the invention. Molecules may bemodified in many ways, including chemically, structurally, andfunctionally. Cells may be modified through the introduction of nucleicacids.

By the term “modulating,” as used herein, is meant mediating adetectable increase or decrease in the level of a response in a subjectcompared with the level of a response in the subject in the absence of atreatment or compound, and/or compared with the level of a response inan otherwise identical but untreated subject. The term encompassesperturbing and/or affecting a native signal or response therebymediating a beneficial therapeutic response in a subject, preferably, ahuman.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

The term “operably linked” refers to functional linkage between aregulatory sequence and a heterologous nucleic acid sequence resultingin expression of the latter. For example, a first nucleic acid sequenceis operably linked with a second nucleic acid sequence when the firstnucleic acid sequence is placed in a functional relationship with thesecond nucleic acid sequence. For instance, a promoter is operablylinked to a coding sequence if the promoter affects the transcription orexpression of the coding sequence. Generally, operably linked DNAsequences are contiguous and, where necessary to join two protein codingregions, in the same reading frame.

The term “overexpressed” tumor antigen or “overexpression” of a tumorantigen is intended to indicate an abnormal level of expression of atumor antigen in a cell from a disease area like a solid tumor within aspecific tissue or organ of the patient relative to the level ofexpression in a normal cell from that tissue or organ. Patients havingsolid tumors or a hematological malignancy characterized byoverexpression of the tumor antigen can be determined by standard assaysknown in the art.

“Parenteral” administration of an immunogenic composition includes,e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR™, and thelike, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

The term “promoter” as used herein is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa polynucleotide sequence.

A “signal transduction pathway” refers to the biochemical relationshipbetween a variety of signal transduction molecules that play a role inthe transmission of a signal from one portion of a cell to anotherportion of a cell. The phrase “cell surface receptor” includes moleculesand complexes of molecules capable of receiving a signal andtransmitting signal across the plasma membrane of a cell.

By the term “specifically binds,” as used herein with respect to anantibody, is meant an antibody which recognizes a specific antigen, butdoes not substantially recognize or bind other molecules in a sample.For example, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. But,such cross-species reactivity does not itself alter the classificationof an antibody as specific. In another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen. However, such cross reactivity does not itselfalter the classification of an antibody as specific. In some instances,the terms “specific binding” or “specifically binding,” can be used inreference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, to mean that the interaction isdependent upon the presence of a particular structure (e.g., anantigenic determinant or epitope) on the chemical species; for example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

By the term “stimulation,” is meant a primary response induced bybinding of a stimulatory molecule (e.g., a TCR/CD3 complex) with itscognate ligand thereby mediating a signal transduction event, such as,but not limited to, signal transduction via the TCR/CD3 complex.Stimulation can mediate altered expression of certain molecules, such asdownregulation of TGF-beta, and/or reorganization of cytoskeletalstructures, and the like.

A “stimulatory molecule,” as the term is used herein, means a moleculeon a T cell that specifically binds with a cognate stimulatory ligandpresent on an antigen presenting cell.

A “stimulatory ligand,” as used herein, means a ligand that when presenton an antigen presenting cell (e.g., an aAPC, a dendritic cell, aB-cell, and the like) can specifically bind with a cognate bindingpartner (referred to herein as a “stimulatory molecule”) on a T cell,thereby mediating a primary response by the T cell, including, but notlimited to, activation, initiation of an immune response, proliferation,and the like. Stimulatory ligands are well-known in the art andencompass, inter alia, an MHC Class I molecule loaded with a peptide, ananti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonistanti-CD2 antibody.

The term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals). A “subject” or“patient,” as used therein, may be a human or non-human mammal.Non-human mammals include, for example, livestock and pets, such asovine, bovine, porcine, canine, feline and murine mammals. Preferably,the subject is human.

As used herein, a “substantially purified” cell is a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cell that have been separated from the cells with which theyare naturally associated in their natural state. In some embodiments,the cells are cultured in vitro. In other embodiments, the cells are notcultured in vitro.

A “target site” or “target sequence” refers to a genomic nucleic acidsequence that defines a portion of a nucleic acid to which a bindingmolecule may specifically bind under conditions sufficient for bindingto occur.

As used herein, the term “T cell receptor” or “TCR” refers to a complexof membrane proteins that participate in the activation of T cells inresponse to the presentation of antigen. The TCR is responsible forrecognizing antigens bound to major histocompatibility complexmolecules. TCR is composed of a heterodimer of an alpha (a) and beta (β)chain, although in some cells the TCR consists of gamma and delta (γ/δ)chains. TCRs may exist in alpha/beta and gamma/delta forms, which arestructurally similar but have distinct anatomical locations andfunctions. Each chain is composed of two extracellular domains, avariable and constant domain. In some embodiments, the TCR may bemodified on any cell comprising a TCR, including, for example, a helperT cell, a cytotoxic T cell, a memory T cell, regulatory T cell, naturalkiller T cell, and gamma delta T cell.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

The phrase “under transcriptional control” or “operatively linked” asused herein means that the promoter is in the correct location andorientation in relation to a polynucleotide to control the initiation oftranscription by RNA polymerase and expression of the polynucleotide.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to, Sendaiviral vectors, adenoviral vectors, adeno-associated virus vectors,retroviral vectors, lentiviral vectors, and the like.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Description

This disclosure describes three chimeric antigen receptors (CARs) thattarget T cell neoplasms as well as a method to prevent fratricide ofhealthy T cells. T cell lymphomas and leukemias are aggressive neoplasmsderived from T cell progenitors or differentiated T cells. Mature orperipheral T-cell lymphomas account for 10%45% of all non-Hodgkin'slymphomas, or ˜7,000-10,000 cases in the U.S/yr. T cell lymphomas andleukemias have poor prognoses and there are few available treatments.CART therapy has demonstrated efficacy for B-cell neoplasms, but untilnow, extending the success of chimeric antigen receptor (CAR) T cells toT-cell malignancies has been problematic because most target antigensare shared between normal and malignant cells, leading to CAR T cellfratricide. Herein CRISPR-Cas editing is used to remove the targetantigen from healthy T cells, protecting them from CART cell therapy andeliminating the potentially fatal immunosuppression that would resultfrom elimination of the T-cell compartment.

In certain embodiments, the CARs target the T cell antigens CD2, CD5 andCD7. In certain embodiments, CRISPR-Cas knock-out of the CD2, CD5, orCD7 target in healthy T cells prevents the killing of healthy T cellsduring manufacturing and subsequent CART therapy.

Methods of Treatment

The present invention includes a method for treating a T cell lymphomaor T cell leukemia in a subject in need thereof. In another aspect, theinvention includes a method for preventing CAR T cell fratricide in asubject in need thereof.

In certain embodiments, the method comprises administering to thesubject a first modified cell comprising a chimeric antigen receptor(CAR), wherein the CAR comprises an antigen binding domain that targetsCD2, a transmembrane domain, and an intracellular domain, andadministering to the subject a second modified cell wherein theendogenous CD2 gene has been knocked-out.

In certain embodiments, the method comprises administering to thesubject a first modified cell comprising a CAR, wherein the CARcomprises an antigen binding domain that targets CD5, a transmembranedomain, and an intracellular domain, and administering to the subject asecond modified cell wherein the endogenous CD5 gene has beenknocked-out.

In certain embodiments, the method comprises administering to thesubject a first modified cell comprising a CAR, wherein the CARcomprises an antigen binding domain that targets CD7, a transmembranedomain, and an intracellular domain, and administering to the subject asecond modified cell wherein the endogenous CD7 gene has beenknocked-out.

In certain embodiments, the method comprises administering to thesubject a first modified cell comprising a chimeric antigen receptor(CAR), wherein the CAR comprises an antigen binding domain, atransmembrane domain, and an intracellular domain, and administering tothe subject a second modified cell wherein the endogenous CD5 gene hasbeen knocked-out.

In certain embodiments, the method comprises administering to thesubject a modified cell comprising a chimeric antigen receptor (CAR),wherein the CAR comprises an antigen binding domain, a transmembranedomain, and an intracellular domain, and wherein the endogenous CD5 genehas been knocked-out in the cell.

In the various embodiments of the methods disclosed herein, the subjectcan be administered any of the CARs disclosed herein. The CAR can bespecific for any tumor associated antigen (TAA) or tumor specificantigen (TSA) known to one of ordinary skill in the art.

In certain embodiments, the CAR comprises a complementarity determiningregion (CDR) comprising the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 31-36, 43-48, 53-58, 65-70, 83-88, and 95-100.In certain embodiments, the CAR comprises an antigen binding domaincomprising a heavy chain variable region comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 29, 41, 51,63, 75, 81, and 93 and/or a light chain variable region comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:30, 42, 52, 64, 76, 82, and 94. In certain embodiments, the CARcomprises an scFv comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 27, 28, 39, 40, 50, 61, 62, 73, 74, 79,80, 91, and 92.

In certain embodiments, the subject is administered a CAR, wherein theCAR comprises a nucleic acid sequence encoded by any one of SEQ ID NOs:1-13. In certain embodiments, the CAR comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 25, 26, 37, 38, 49,59, 60, 71, 72, 77, 78, 89, and 90.

In certain embodiments, the first and or second modified cell is a Tcell. In certain embodiments, the cancer comprises a T cell lymphoma ora T cell leukemia. Types of cancers that can be treated with thecompositions and methods of the present invention include but are notlimited to non-Hodgkins's lymphoma and subtypes thereof includingperipheral T-cell lymphoma (PTCL), Angioimmunoblastic T-cell lymphoma(AITL), anaplastic lymphoma kinase (ALK)-negative anaplastic large celllymphoma (ALCL, ALK−), Natural killer/T-cell lymphoma (NKTCL), AdultT-cell leukemia/lymphoma (ATLL), ALCL ALK+, enteropathy-type T-cell,hepatosplenic T-cell, subcutaneous panniculitis-like, and unclassifiablePTCL.

In certain embodiments, the endogenous gene (e.g. CD2, CD5, and CD7) isknocked-out using a CRISPR/Cas9 method. In certain embodiments, theCRISPR/Cas9 method utilizes an sgRNA targeting CD2, CD5, and/or CD7. Incertain embodiments, the sgRNA comprises the nucleotide sequenceselected from the group consisting of SEQ ID NOs: 22-24.

In certain embodiments, the CAR of present invention further comprises asuicide gene. One non-limiting example of a suicide gene is theinducible caspase 9 gene (iCaspase9, iCasp9 or iC9). The iCaspase9suicide gene system is based on the fusion of human caspase 9 to amodified human FK-binding protein, allowing conditional dimerizationusing a small-molecule drug (e.g. AP1903). When exposed to a syntheticdimerizing drug, the iCaspase9 becomes activated and leads to the rapidapoptosis of cells expressing this construct (e.g. the CART cell) (Zhouet al. (2015) Methods Mol Biol. 1317: 87-105). Another example of asuicide gene is the HSV-tk gene (Bordingnon et al. (1995) Human GeneTherapy, vol. 6, no. 6, pp 813-819). The HSV-tk gene can be co-expressedin the CAR T cell, and upon expression it turns the non-toxic prodrugGCV into GCV-triphosphate, leading to cell death by halting DNAreplication.

Compositions

One aspect of the invention includes a composition comprising a firstmodified cell comprising a chimeric antigen receptor (CAR), wherein theCAR comprises an antigen binding domain that targets CD2, atransmembrane domain, and an intracellular domain, and a second modifiedcell wherein the endogenous CD2 gene has been knocked-out.

Another aspect of the invention includes a composition comprising afirst modified cell comprising a CAR, wherein the CAR comprises anantigen binding domain that targets CD5, a transmembrane domain, and anintracellular domain, and a second modified cell wherein the endogenousCD5 gene has been knocked-out.

Yet another aspect of the invention includes a composition comprising afirst modified cell comprising a CAR, wherein the CAR comprises anantigen binding domain that targets CD7, a transmembrane domain, and anintracellular domain, and a second modified cell wherein the endogenousCD7 gene has been knocked-out.

In certain embodiments, the CAR is encoded by a nucleic acid sequenceselected from the group consisting of SEQ ID NOs: 1-13. In certainembodiments, the endogenous genes have been knocked-out using aCRISPR/Cas system. In certain embodiments, the CRISPR/Cas9 systemcomprises a gRNA comprising a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 22-24.

The invention further includes the composition of the invention and apharmaceutically acceptable carrier.

Chimeric Antigen Receptor (CAR)

The present invention provides a chimeric antigen receptor (CAR)comprising an antigen binding domain, a transmembrane domain, and anintracellular domain. In certain embodiments, the invention comprises aCAR comprising an antigen binding domain capable of binding CD2, atransmembrane domain, and an intracellular domain.

Antigen Binding Domain

In one embodiment, the CAR of the invention comprises a target-specificbinding element otherwise referred to as an antigen binding domain. Thechoice of antigen binding domain depends upon the type and number ofligands that define the surface of a target cell. For example, theantigen binding domain may be chosen to recognize a ligand that acts asa cell surface marker on target cells associated with a particulardisease state (e.g. T cell lymphoma or leukemia).

In one embodiment, the CAR of the invention can be engineered to targeta tumor antigen. The antigens discussed herein are merely included byway of example. The list is not intended to be exclusive and furtherexamples will be readily apparent to those of skill in the art. Tumorantigens are proteins that are produced by tumor cells that elicit animmune response, particularly T-cell mediated immune responses. Theselection of the antigen binding domain of the invention will depend onthe particular type of cancer to be treated. Tumor antigens are wellknown in the art and include, for example, a glioma-associated antigen,carcinoembryonic antigen (CEA), β-human chorionic gonadotropin,alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1,MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS),intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase,prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein,PSMA, Her2/neu, survivin and telomerase, prostate-carcinoma tumorantigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22,insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

The type of tumor antigen referred to in the invention may also be atumor-specific antigen (TSA) or a tumor-associated antigen (TAA). A TSAis unique to tumor cells and does not occur on other cells in the body.A TAA associated antigen is not unique to a tumor cell and instead isalso expressed on a normal cell under conditions that fail to induce astate of immunologic tolerance to the antigen. The expression of theantigen on the tumor may occur under conditions that enable the immunesystem to respond to the antigen. TAAs may be antigens that areexpressed on normal cells during fetal development when the immunesystem is immature and unable to respond or they may be antigens thatare normally present at extremely low levels on normal cells but whichare expressed at much higher levels on tumor cells.

Non-limiting examples of TSA or TAA antigens include the following:Differentiation antigens such as MART-1/MelanA (MART-I), gplOO (Pmel17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigenssuch as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressedembryonic antigens such as CEA; overexpressed oncogenes and mutatedtumor-suppressor genes such as p53, Ras, HER-2/neu; unique tumorantigens resulting from chromosomal translocations; such as BCR-ABL,E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as theEpstein Barr virus antigens EBVA and the human papillomavirus (HPV)antigens E6 and E7. Other large, protein-based antigens include TSP-180,MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met,nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras,beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72,alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250,Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1,RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associatedprotein, TAAL6, TAG72, TLP, and TPS.

Depending on the desired antigen to be targeted, the CAR of theinvention can be engineered to include the appropriate antigen bindingdomain that is specific to the desired antigen target. For example, ifCD2 is the desired antigen that is to be targeted, an antibody for CD2can be used as the antigen binding domain for incorporation into the CARof the invention.

In certain embodiments, the antigen binding domain of the CAR targetsCD2. In certain embodiments, the antigen binding domain of the CARtargets CD5. In certain embodiments, the antigen binding domain of theCAR targets CD7.

In some embodiments, the antigen binding domain in the CAR of theinvention is anti-CD2 scFV. In some embodiments, the antigen bindingdomain in the CAR of the invention is anti-CD5 scFV. In someembodiments, the antigen binding domain in the CAR of the invention isanti-CD7 scFV. In some embodiments, the antigen binding domain is ananti-CD2 antibody. In some embodiments, the antigen binding domain is ananti-CD5 antibody. In some embodiments, the antigen binding domain is ananti-CD7 antibody.

In certain embodiments, the antigen binding domain comprises a heavychain variable region that comprises three heavy chain complementaritydetermining regions (HCDRs) and a light chain variable region thatcomprises three light chain complementarity determining regions (LCDRs).

In certain embodiments, the invention comprises a CAR comprising anantigen binding domain capable of binding CD2, wherein the antigenbinding domain comprises a complementarity determining region (CDR)comprising the amino acid sequence of any one of SEQ ID NOs: 31, 32, 33,34, 35, 36, 43, 44, 45, 46, 47, 48, 53, 54, 55, 56, 57, 58, 65, 66, 67,68, 69, or 70.

In certain embodiments, the CAR comprises an antigen binding domaincapable of binding CD2, wherein HCDR1 comprises the amino acid sequenceof SEQ ID NO: 31, HCDR2 comprises the amino acid sequence of SEQ ID NO:32, HCDR3 comprises the amino acid sequence of SEQ ID NO: 33, LCDR1comprises the amino acid sequence of SEQ ID NO: 34, LCDR2 comprises theamino acid sequence of SEQ ID NO: 35, and LCDR3 comprises the amino acidsequence of SEQ ID NO: 36.

In certain embodiments, the CAR comprises an antigen binding domaincapable of binding CD2, wherein HCDR1 comprises the amino acid sequenceof SEQ ID NO: 43, HCDR2 comprises the amino acid sequence of SEQ ID NO:44, HCDR3 comprises the amino acid sequence of SEQ ID NO: 45, LCDR1comprises the amino acid sequence of SEQ ID NO: 46, LCDR2 comprises theamino acid sequence of SEQ ID NO: 47, and LCDR3 comprises the amino acidsequence of SEQ ID NO: 48.

In certain embodiments, the CAR comprises an antigen binding domaincapable of binding CD2, wherein HCDR1 comprises the amino acid sequenceof SEQ ID NO: 65, HCDR2 comprises the amino acid sequence of SEQ ID NO:66, HCDR3 comprises the amino acid sequence of SEQ ID NO: 67, LCDR1comprises the amino acid sequence of SEQ ID NO: 68, LCDR2 comprises theamino acid sequence of SEQ ID NO: 69, and LCDR3 comprises the amino acidsequence of SEQ ID NO: 70.

In certain embodiments, the CAR comprises an antigen binding domaincapable of binding CD2, wherein the antigen binding domain comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 29 and/or a light chain variable region comprising the amino acidsequence of SEQ ID NO: 30. In certain embodiments, the antigen bindingdomain comprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 41 and/or a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 42. In certainembodiments, the antigen binding domain comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 51 and/or alight chain variable region comprising the amino acid sequence of SEQ IDNO: 52. In certain embodiments, the antigen binding domain comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 63 and/or a light chain variable region comprising the amino acidsequence of SEQ ID NO: 64.

In certain embodiments, the CAR comprises an antigen binding domaincapable of binding CD2, wherein the antigen binding domain is a scFvcomprising the amino acid sequence set forth in any one of SEQ ID NOs:27, 28, 39, 40, 50, 61, or 62.

In certain embodiments, the invention comprises a CAR comprising anantigen binding domain capable of binding CD5, wherein the antigenbinding domain comprises a complementarity determining region (CDR)comprising the amino acid sequence of any one of SEQ ID NOs: 83, 84, 85,86, 87, 88, 95, 96, 97, 98, 99, or 100.

In certain embodiments, the CAR comprises an antigen binding domaincapable of binding CD5, wherein HCDR1 comprises the amino acid sequenceof SEQ ID NO: 83, HCDR2 comprises the amino acid sequence of SEQ ID NO:84, HCDR3 comprises the amino acid sequence of SEQ ID NO: 85, LCDR1comprises the amino acid sequence of SEQ ID NO: 86, LCDR2 comprises theamino acid sequence of SEQ ID NO: 87, and LCDR3 comprises the amino acidsequence of SEQ ID NO: 88.

In certain embodiments, the CAR comprises an antigen binding domaincapable of binding CD5, wherein HCDR1 comprises the amino acid sequenceof SEQ ID NO: 95, HCDR2 comprises the amino acid sequence of SEQ ID NO:96, HCDR3 comprises the amino acid sequence of SEQ ID NO: 97, LCDR1comprises the amino acid sequence of SEQ ID NO: 98, LCDR2 comprises theamino acid sequence of SEQ ID NO: 99, and LCDR3 comprises the amino acidsequence of SEQ ID NO: 100.

In certain embodiments, the CAR comprises an antigen binding domaincapable of binding CD5, wherein the antigen binding domain comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 75 and/or a light chain variable region comprising the amino acidsequence of SEQ ID NO: 76. In certain embodiments, the heavy chainvariable region comprises the amino acid sequence of SEQ ID NO: 81and/or a light chain variable region comprises the amino acid sequenceof SEQ ID NO: 82. In certain embodiments, the heavy chain variableregion comprises the amino acid sequence of SEQ ID NO: 93 and/or a lightchain variable region comprises the amino acid sequence of SEQ ID NO:94.

In certain embodiments, the CAR comprises an antigen binding domaincapable of binding CD5, wherein the antigen binding domain is a scFvcomprising the amino acid sequence set forth in any one of SEQ ID NOs:73, 74, 79, 80, 91, or 92.

Tolerable variations of the antigen binding domain sequences will beknown to those of skill in the art. For example, in some embodiments theantigen binding domain comprises an amino acid sequence that has atleast 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to any of the amino acidsequences set forth in SEQ ID NOs: 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 55, 56,57, 58, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 73, 74, 75, 76, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 91, 92, 93, 94, 95, 96, 97, 98, 99, or100.

Transmembrane Domain

With respect to the transmembrane domain, the CAR can be designed tocomprise a transmembrane domain that is fused to the extracellulardomain of the CAR. In one embodiment, the transmembrane domain thatnaturally is associated with one of the domains in the CAR is used. Insome instances, the transmembrane domain can be selected or modified byamino acid substitution to avoid binding of such domains to thetransmembrane domains of the same or different surface membrane proteinsto minimize interactions with other members of the receptor complex.

The transmembrane domain may be derived either from a natural or from asynthetic source. Where the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein. Transmembrane regionsof particular use in this invention may be derived from (i.e. compriseat least the transmembrane region(s) of) the alpha, beta or zeta chainof the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9,CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.Alternatively the transmembrane domain may be synthetic, in which caseit will comprise predominantly hydrophobic residues such as leucine andvaline. Preferably a triplet of phenylalanine, tryptophan and valinewill be found at each end of a synthetic transmembrane domain.Optionally, a short oligo- or polypeptide linker, preferably between 2and 10 amino acids in length may form the linkage between thetransmembrane domain and the cytoplasmic signaling domain of the CAR. Aglycine-serine doublet provides a particularly suitable linker.

In one embodiment, the transmembrane domain in the CAR of the inventionis a CD8 transmembrane domain. In one embodiment, the CD8 transmembranedomain comprises the nucleic acid sequence of SEQ ID NO: 14. In oneembodiment, the CD8 transmembrane domain comprises the nucleic acidsequence that encodes the amino acid sequence of SEQ ID NO: 15. Inanother embodiment, the CD8 transmembrane domain comprises the aminoacid sequence of SEQ ID NO: 15.

In some instances, the transmembrane domain of the CAR of the inventioncomprises the CD8a hinge domain. In one embodiment, the CD8 hinge domaincomprises the nucleic acid sequence of SEQ ID NO: 16. In one embodiment,the CD8 hinge domain comprises a nucleic acid sequence that encodes theamino acid sequence of SEQ ID NO: 17. In another embodiment, the CD8hinge domain comprises the amino acid sequence of SEQ ID NO: 17.

Between the antigen binding domain and the transmembrane domain of theCAR, or between the intracellular domain and the transmembrane domain ofthe CAR, there may be incorporated a spacer domain. As used herein, theterm “spacer domain” generally means any oligo- or polypeptide thatfunctions to link the transmembrane domain to, either the extracellulardomain or, the cytoplasmic domain in the polypeptide chain. A spacerdomain may comprise up to 300 amino acids, preferably 10 to 100 aminoacids and most preferably 25 to 50 amino acids.

Intracellular Domain

The intracellular domain or otherwise the cytoplasmic domain of the CARof the invention is responsible for activation of at least one of thenormal effector functions of the immune cell in which the CAR has beenplaced in. The term “effector function” refers to a specialized functionof a cell. Effector function of a T cell, for example, may be cytolyticactivity or helper activity including the secretion of cytokines. Thusthe term “intracellular domain” refers to the portion of a protein whichtransduces the effector function signal and directs the cell to performa specialized function. While usually the entire intracellular domaincan be employed, in many cases it is not necessary to use the entirechain. To the extent that a truncated portion of the intracellulardomain is used, such truncated portion may be used in place of theintact chain as long as it transduces the effector function signal. Theterm intracellular domain is thus meant to include any truncated portionof the intracellular domain sufficient to transduce the effectorfunction signal.

Preferred examples of intracellular domains for use in the CAR of theinvention include the cytoplasmic sequences of the T cell receptor (TCR)and co-receptors that act in concert to initiate signal transductionfollowing antigen receptor engagement, as well as any derivative orvariant of these sequences and any synthetic sequence that has the samefunctional capability.

It is known that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondary orco-stimulatory signal is also required. Thus, T cell activation can besaid to be mediated by two distinct classes of cytoplasmic signalingsequence: those that initiate antigen-dependent primary activationthrough the TCR (primary cytoplasmic signaling sequences) and those thatact in an antigen-independent manner to provide a secondary orco-stimulatory signal (secondary cytoplasmic signaling sequences).

Primary cytoplasmic signaling sequences regulate primary activation ofthe TCR complex either in a stimulatory way, or in an inhibitory way.Primary cytoplasmic signaling sequences that act in a stimulatory mannermay contain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs.

Examples of ITAM containing primary cytoplasmic signaling sequences thatare of particular use in the invention include those derived from TCRzeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22,CD79a, CD79b, and CD66d. It is particularly preferred that cytoplasmicsignaling molecule in the CAR of the invention comprises a cytoplasmicsignaling sequence derived from CD3 zeta.

In a preferred embodiment, the intracellular domain of the CAR can bedesigned to comprise the CD3-zeta signaling domain by itself or combinedwith any other desired intracellular domain(s) useful in the context ofthe CAR of the invention. For example, the intracellular domain of theCAR can comprise a CD3 zeta chain portion and a costimulatory signalingregion. The costimulatory signaling region refers to a portion of theCAR comprising the intracellular domain of a costimulatory molecule. Acostimulatory molecule is a cell surface molecule other than an antigenreceptor or their ligands that is required for an efficient response oflymphocytes to an antigen. Examples of such molecules include, but arenot limited to, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS,lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, B7-H3, and a ligand that specifically binds with CD83, and thelike. Thus, while the invention is exemplified primarily with 4-1BB asthe co-stimulatory signaling element, other costimulatory elements arewithin the scope of the invention.

The cytoplasmic signaling sequences within the cytoplasmic signalingportion of the CAR of the invention may be linked to each other in arandom or specified order. Optionally, a short oligo- or polypeptidelinker, preferably between 2 and 10 amino acids in length may form thelinkage. A glycine-serine doublet provides a particularly suitablelinker.

In one embodiment, the intracellular domain is designed to comprise thesignaling domain of CD3-zeta and the signaling domain of CD28. Inanother embodiment, the intracellular domain is designed to comprise thesignaling domain of CD3-zeta and the signaling domain of 4-1BB. In yetanother embodiment, the intracellular domain is designed to comprise thesignaling domain of CD3-zeta and the signaling domain of CD28 and 4-1BB.

In one embodiment, the intracellular domain in the CAR of the inventionis designed to comprise the signaling domain of 4-1BB and the signalingdomain of CD3-zeta, wherein the signaling domain of 4-1BB comprises thenucleic acid sequence set forth in SEQ ID NO: 18 and the signalingdomain of CD3-zeta comprises the nucleic acid sequence set forth in SEQID NO: 19.

In one embodiment, the intracellular domain in the CAR of the inventionis designed to comprise the signaling domain of 4-1BB and the signalingdomain of CD3-zeta, wherein the signaling domain of 4-1BB comprises anucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:20 and the signaling domain of CD3-zeta comprises a nucleic acidsequence that encodes the amino acid sequence of SEQ ID NO: 21.

In one embodiment, the intracellular domain in the CAR of the inventionis designed to comprise the signaling domain of 4-1BB and the signalingdomain of CD3-zeta, wherein the signaling domain of 4-1BB comprises theamino acid sequence set forth in SEQ ID NO: 20 and the signaling domainof CD3-zeta comprises the amino acid sequence set forth in SEQ ID NO:21.

In one embodiment, the anti-CD2 CAR comprises the amino acid sequenceset forth in any one of SEQ ID NOs: 25, 26, 37, 38, 49, 59, or 60. Inone embodiment, the anti-CD2 CAR is encoded by a nucleic acid sequenceselected from the group consisting of SEQ ID NOs: 1-7. In oneembodiment, the anti-CD5 CAR comprises the amino acid sequence set forthin any one of SEQ ID NOs: 71, 72, 77, 78, 89, or 90. In one embodiment,the anti-CD5 CAR is encoded by a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 8-13.

Tolerable variations of the CAR sequences will be known to those ofskill in the art. For example, in some embodiments the CAR comprises anamino acid sequence that has at least 70%, at least 75%, at least 80%,at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% sequence identityto any of the amino acid sequences set forth in SEQ ID NO: 25, 26, 37,38, 49, 59, 60, 71, 72, 77, 78, 89, or 90. In some embodiments the CARis encoded by a nucleic acid sequence that has at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least or 99% sequence identity to the nucleic acidsequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,or 13.

The invention should be construed to include any one of: a CAR, anucleic acid encoding a CAR, a vector comprising a nucleic acid encodinga CAR, a cell comprising a CAR, a cell comprising a nucleic acidencoding a CAR, and a cell comprising a vector comprising a nucleic acidencoding a CAR.

CD2-MEDI507H2L-3028 CAR (SEQ ID NO: 1)ggatccCAAGTCCAACTGGTGCAATCAGGCGCAGAAGTCCAACGACCGGGGGCCAGTGTTAAAGTGTCTTGTAAAGCCTCCGGGTACATTTTTACTGAGTACTATATGTACTGGGTCAGACAGGCCCCAGGGCAAGGTTTGGAACTTGTCGGACGCATAGATCCCGAAGACGGTTCTATAGATTACGTTGAGAAGTTCAAAAAGAAAGTCACACTTACTGCGGACACATCTAGTAGCACCGCATATATGGAACTGAGCAGTCTCACCTCAGACGACACCGCAGTGTACTATTGCGCTCGCGGAAAGTTTAACTATAGGTTCGCGTACTGGGGACAGGGGACACTGGTGACTGTTAGCAGCggtggcggagggagcggcggtggaggaagcggaggcggaggttccGACGTTGTGATGACGCAAAGTCCCCCGTCACTCCTTGTTACTCTCGGCCAGCCAGCGTCTATCTCTTGCCGGTCAAGCCAGAGCTTGCTCCACTCTAGTGGTAACACGTATTTGAACTGGTTGCTGCAAAGGCCTGGACAATCTCCTCAGCCCCTGATCTATTTGGTTAGCAAACTGGAAAGTGGTGTTCCAGACAGATTTTCAGGGTCTGGATCAGGCACTGATTTCACTCTGAAGATCTCCGGGGTAGAGGCCGAGGACGTGGGAGTCTATTACTGCATGCAGTTTACTCACTATCCTTATACCTTTGGTCAAGGGACGAAACTGGAGATCAAAtccgga CD2-OKT11H2L-3029 CAR (SEQ ID NO: 2)ggatccCAAGTTCAGCTTCAGCAACCAGGTGCTGAATTGGTCCGCCCTGGAACTAGCGTTAAACTGTCTTGTAAGGCATCCGGTTATACGTTTACAAGTTATTGGATGCACTGGATTAAGCAAAGGCCCGAACAAGGCCTTGAATGGATTGGGAGAATTGATCCCTACGATAGCGAGACACACTACAATGAAAAATTTAAAGATAAGGCCATCCTCAGCGTAGATAAGAGCAGTTCTACCGCATACATACAGCTCTCAAGCCTGACGTCAGATGACTCAGCCGTTTATTATTGCTCAAGGCGGGACGCTAAATACGACGGCTATGCGCTTGACTACTGGGGACAAGGCACCACTTTGACAGTCTCCAGTggtggcggagggagcggcggtggaggaagcggaggcggaggttccGATATAGTTATGACGCAAGCAGCACCCTCTGTACCTGTGACACCGGGTGAATCCGTTAGTATCTCATGCCGCTCTTCTAAAACCCTCTTGCATTCTAACGGCAATACATATTTGTATTGGTTCCTTCAACGACCAGGACAATCACCGCAAGTGCTTATTTATAGGATGTCTAACTTGGCTAGTGGGGTGCCAAATAGGTTCAGTGGGTCTGGATCTGAGACAACTTTCACGTTGAGAATAAGTAGGGTGGAAGCTGAAGACGTCGGTATATACTACTGTATGCAGCATTTGGAGTACCCTTACACTTTCGGGGGAGGTACTAAGCTCGAAATTAAAtccgga CD2-OKT11L2H-3030 CAR  (SEQ ID NO: 3)ggatccGATATAGTTATGACGCAAGCAGCACCCTCTGTACCTGTGACACCGGGTGAATCCGTTAGTATCTCATGCCGCTCTTCTAAAACCCTCTTGCATTCTAACGGCAATACATATTTGTATTGGTTCCTTCAACGACCAGGACAATCACCGCAAGTGCTTATTTATAGGATGTCTAACTTGGCTAGTGGGGTGCCAAATAGGTTCAGTGGGTCTGGATCTGAGACAACTTTCACGTTGAGAATAAGTAGGGTGGAAGCTGAAGACGTCGGTATATACTACTGTATGCAGCATTTGGAGTACCCTTACACTTTCGGGGGAGGTACTAAGCTCGAAATTAAAggtggcggagggagcggcggtggaggaagcggaggcggaggttccCAAGTTCAGCTTCAGCAACCAGGTGCTGAATTGGTCCGCCCTGGAACTAGCGTTAAACTGTCTTGTAAGGCATCCGGTTATACGTTTACAAGTTATTGGATGCACTGGATTAAGCAAAGGCCCGAACAAGGCCTTGAATGGATTGGGAGAATTGATCCCTACGATAGCGAGACACACTACAATGAAAAATTTAAAGATAAGGCCATCCTCAGCGTAGATAAGAGCAGTTCTACCGCATACATACAGCTCTCAAGCCTGACGTCAGATGACTCAGCCGTTTATTATTGCTCAAGGCGGGACGCTAAATACGACGGCTATGCGCTTGACTACTGGGGACAAGGCACCACTTTGACAGTCTCCAGTtccgga CD2-T11-2-H2L-3031 CAR (SEQ ID NO: 4)ggatccCAAGTTCAATTGCAGCAACCGGGTGCCGAGTTGGTAAGGCCCGGTGCGTCAGTCAAACTTAGTTGTAAAGCTAGTGGGTACACTTTTACTACGTTCTGGATGAATTGGGTGAAGCAACGACCAGGCCAAGGTCTGGAATGGATCGGCATGATTGACCCGTCTGACTCAGAAGCTCATTACAACCAGATGTTCAAGGACAAGGCGACTCTGACTGTTGATAAAAGCTCAAGCACCGCCTACATGCAGCTCAGTAGCCTCACATCCGAGGATTCCGCAGTGTACTATTGCGCGAGGGGACGAGGGTATGATGACGGCGATGCGATGGACTATTGGGGACAGGGGACCAGCGTAACAGTCAGTAGTggtggcggagggagcggcggtggaggaagcggaggcggaggttccGATATAGTTATGACCCAGTCTCCCGCCTCTCTGGCCGTTAGCTTGGGACAACGCGCTACCATCTCTTACCGAGCGTCTAAGTCCGTCAGTACAAGCGGTTATAGTTACATGCACTGGAACCAGCAAAAGCCCGGACAACCTCCGAGACTCCTGATTTATTTGGTCTCTAACCTTGAGTCAGGTGTCCCAGCCAGATTCTCCGGCTCTGGAAGCGGCACTGACTTTACATTGAACATTCACCCCGTGGAGGAGGAAGACGCTGCTACCTACTATTGCATGCAATTCACGCACTATCCCTACACATTCGGGGGGGGCACGAAATTGGAAATCAAAtccgga CD2-TS2-18.1.1-H2L-3032 CAR (SEQ ID NO: 5)ggatccGAGGTTCAGCTTGAGGAGAGTGGGGGAGGTTTGGTAATGCCAGGTGGGTCTTTGAAACTCAGTTGCGCGGCGTCAGGCTTCGCATTTTCCTCCTACGATATGTCCTGGGTCAGACAGACACCCGAGAAGCGGCTGGAATGGGTCGCTTACATTTCCGGGGGAGGATTCACGTACTACCCGGATACAGTAAAGGGGAGATTTACTCTGAGCCGGGACAACGCTAAGAATACCCTCTATCTCCAGATGTCCTCTTTGAAGAGTGAAGACACAGCGATGTATTACTGTGCGAGACAAGGGGCCAATTGGGAGCTGGTTTACTGGGGCCAGGGGACGACATTGACGGTTTCTAGCggtggcggagggagcggcggtggaggaagcggaggcggaggttccGACATTGTAATGACACAATCACCTGCTACACTTAGCGTGACTCCAGGTGATCGGGTATTCCTGAGCTGCCGCGCATCACAAAGTATATCCGACTTCCTGCACTGGTATCAGCAGAAATCTCACGAAAGTCCCAGGCTGCTGATTAAATACGCTTCCCAGAGTATTAGTGGTATCCCCTCACGATTTTCTGGCAGCGGGAGCGGTAGTGACTTCACTCTTTCTATAAACTCCGTCGAGCCAGAAGACGTGGGGGTGTATCTTTGCCAAAATGGACACAATTTTCCACCAACCTTTGGTGGGGGCACCAAACTCGAAATAAAGtccg gaCD2-TS2-18.1.1-L2H-3033 CAR (SEQ ID NO: 6)ggatccGACATTGTAATGACACAATCACCTGCTACACTTAGCGTGACTCCAGGTGATCGGGTATTCCTGAGCTGCCGCGCATCACAAAGTATATCCGACTTCCTGCACTGGTATCAGCAGAAATCTCACGAAAGTCCCAGGCTGCTGATTAAATACGCTTCCCAGAGTATTAGTGGTATCCCCTCACGATTTTCTGGCAGCGGGAGCGGTAGTGACTTCACTCTTTCTATAAACTCCGTCGAGCCAGAAGACGTGGGGGTGTATCTTTGCCAAAATGGACACAATTTTCCACCAACCTTTGGTGGGGGCACCAAACTCGAAATAAAGggtggcggagggagcggcggtggaggaagcggaggcggaggttccGAGGTTCAGCTTGAGGAGAGTGGGGGAGGTTTGGTAATGCCAGGTGGGTCTTTGAAACTCAGTTGCGCGGCGTCAGGCTTCGCATTTTCCTCCTACGATATGTCCTGGGTCAGACAGACACCCGAGAAGCGGCTGGAATGGGTCGCTTACATTTCCGGGGGAGGATTCACGTACTACCCGGATACAGTAAAGGGGAGATTTACTCTGAGCCGGGACAACGCTAAGAATACCCTCTATCTCCAGATGTCCTCTTTGAAGAGTGAAGACACAGCGATGTATTACTGTGCGAGACAAGGGGCCAATTGGGAGCTGGTTTACTGGGGCCAGGGGACGACATTGACGGTTTCTAGCtcc ggaCD2-MEDI507L2H-3043 CAR (SEQ ID NO: 7)ggatccGACGTTGTGATGACGCAAAGTCCCCCGTCACTCCTTGTTACTCTCGGCCAGCCAGCGTCTATCTCTTGCCGGTCAAGCCAGAGCTTGCTCCACTCTAGTGGTAACACGTATTTGAACTGGTTGCTGCAAAGGCCTGGACAATCTCCTCAGCCCCTGATCTATTTGGTTAGCAAACTGGAAAGTGGTGTTCCAGACAGATTTTCAGGGTCTGGATCAGGCACTGATTTCACTCTGAAGATCTCCGGGGTAGAGGCCGAGGACGTGGGAGTCTATTACTGCATGCAGTTTACTCACTATCCTTATACCTTTGGTCAAGGGACGAAACTGGAGATCAAAggtggcggagggagcggcggtggaggaagcggaggcggaggttccCAAGTCCAACTGGTGCAATCAGGCGCAGAAGTCCAACGACCGGGGGCCAGTGTTAAAGTGTCTTGTAAAGCCTCCGGGTACATTTTTACTGAGTACTATATGTACTGGGTCAGACAGGCCCCAGGGCAAGGTTTGGAACTTGTCGGACGCATAGATCCCGAAGACGGTTCTATAGATTACGTTGAGAAGTTCAAAAAGAAAGTCACACTTACTGCGGACACATCTAGTAGCACCGCATATATGGAACTGAGCAGTCTCACCTCAGACGACACCGCAGTGTACTATTGCGCTCGCGGAAAGTTTAACTATAGGTTCGCGTACTGGGGACAGGGGACACTGGTGACTGTTAGCAGCtccgga CD5-17L2H-3045 CAR (SEQ ID NO: 8)ggatccAACATTGTACTGACGCAAAGCCCCTCATCTTTGTCTGAGTCACTCGGCGGCAAAGTAACCATCACATGCAAGGCCAGTCAAGACATCAATAAATATATTGCTTGGTATCAGTATAAACCCGGCAAGGGGCCGCGACTGCTGATTCACTACACGAGTACCTTGCAACCGGGCATTCCGAGCCGATTTAGTGGCAGTGGCTCAGGTCGCGATTACTCATTCTCAATAAGTAATCTCGAACCGGAAGACATAGCTACTTATTATTGCTTGCAGTACGATAATTTGTGGACCTTCGGGGGTGGTACAAAGTTGGAAATAAAGggtggcggagggagcggcggtggaggaagcggaggcggaggttccGAGGTCCAACTCGTAGAATCAGGTCCCGGATTGGTGCAACCATCCCAGAGCCTCTCTATTACATGCACGGTCTCTGGATTTAGTCTGACCAATTACGATGTGCATTGGGTGCGCCAGTCTCCCGGCAAGGGGTTGGAATGGCTTGGCGTTATATGGAACTACGGAAATACAGACTATAACGCCGCGTTTATCTCTCGGCTGAGTATACGGAAAGACAGTAGTAAATCCCAGGTCTTTTTTACGATGTCATCCCTGCAAACGCCAGATACCGCAATATATTACTGCGCCAGGAACCACGGTGATGGTTATTATAATTGGTACTTCGATGTGTGGGGTACTGGCACTACAGTCACAGTA TCTTCAtctagaCD5-9H2L-3048 CAR (SEQ ID NO: 9)ggatcc CAG GTC CAG CTG AAA GAA AGC GGT CCA GAG CTG GAA AAA CCCGGT GCG AGC GTC AAA ATA TCA TGT AAA GCA AGC GGG TAT TCA TTC ACCGCG TAC TCT ATG AAC TGG GTT AAG CAA AAC AAC GGT ATG TCC TTG GAGTGG ATA GGG TCT ATC GAC CCG TAT TAT GGG GAC ACA AAA TAC GCG CAGAAA TTC AAG GGG AAG GCC ACC CTG ACC GTA GAT AAA GCT AGT TCT ACTGCG TAC TTG CAA CTG AAA AGC CTC ACT TCT GAG GAC TCT GCC GTC TACTAC TGT GCT CGG CGA ATG ATA ACG ACG GGG GAC TGG TAT TTC GAT GTTTGG GGT ACA GGG ACT ACG GTG ACT GTC AGTAGCggtggcggagggagcggcggtggaggaagcggaggcggaggttcc CAT ATC GTC TTG ACT CAATCA CCT AGT TCT TTG TCT GCG TCC CTT GGC GAC CGA GTC ACC ATA TCTTGC AGA GCG TCA CAG GAC ATT TCA ACG TAC CTC AAC TGG TAT CAG CAAAAA CCG GAC GGG ACT GTC AAG CTC TTG ATC TTC TAC ACT TCC AGA CTCCAC GCC GGG GTG CCA AGC AGA TTT AGT GGC TCT GGC AGC GGG ACA CACCAT AGT CTT ACA ATC AGC AAT CTT GAG CAA GAA GAC ATA GCC ACG TATTTC TGC CAG CAA GGT AAC TCA CTT CCG TTC ACG TTT GGT AGT GGC ACCAAA CTG GAG ATA AAA tccgga CD5-9L2H-3049 CAR (SEQ ID NO: 10)Ggatcc CAT ATC GTC TTG ACT CAA TCA CCT AGT TCT TTG TCT GCG TCC CTTGGC GAC CGA GTC ACC ATA TCT TGC AGA GCG TCA CAG GAC ATT TCA ACGTAC CTC AAC TGG TAT CAG CAA AAA CCG GAC GGG ACT GTC AAG CTC TTGATC TTC TAC ACT TCC AGA CTC CAC GCC GGG GTG CCA AGC AGA TTT AGTGGC TCT GGC AGC GGG ACA CAC CAT AGT CTT ACA ATC AGC AAT CTT GAGCAA GAA GAC ATA GCC ACG TAT TTC TGC CAG CAA GGT AAC TCA CTT CCGTTC ACG TTT GGT AGT GGC ACC AAA CTG GAG ATA AAAggtggcggagggagcggcggtggaggaagcggaggcggaggttcc CAG GTC CAG CTG AAA GAA AGCGGT CCA GAG CTG GAA AAA CCC GGT GCG AGC GTC AAA ATA TCA TGT AAAGCA AGC GGG TAT TCA TTC ACC GCG TAC TCT ATG AAC TGG GTT AAG CAAAAC AAC GGT ATG TCC TTG GAG TGG ATA GGG TCT ATC GAC CCG TAT TATGGG GAC ACA AAA TAC GCG CAG AAA TTC AAG GGG AAG GCC ACC CTG ACCGTA GAT AAA GCT AGT TCT ACT GCG TAC TTG CAA CTG AAA AGC CTC ACTTCT GAG GAC TCT GCC GTC TAC TAC TGT GCT CGG CGA ATG ATA ACG ACGGGG GAC TGG TAT TTC GAT GTT TGG GGT ACA GGG ACT ACG GTG ACT GTCAGT AGC tccgga CD5-34H2L-3052 CAR (SEQ ID NO: 11)ggatccGAGGTTAAACTCGTGGAGAGCGGTGCCGAACTCGTCCGAAGTGGTGCTTCCGTTAAACTCAGTTGTGCCGCGTCAGGATTTAACATAAAAGATTACTACATTCACTGGGTCAAACAGCGCCCGGAGCAGGGGCTTGAATGGATCGGGTGGATTGATCCTGAAAACGGGCGCACCGAATATGCTCCCAAGTTCCAGGGCAAAGCTACTATGACCGCTGACACCTCTAGTAACACTGCCTACCTGCAGTTGAGCTCTCTTACGTCTGAGGATACCGCTGTGTACTACTGTAATAACGGAAATTATGTACGACACTATTACTTCGACTACTGGGGGCAGGGCACTACTGTGACTGTATCTAGCggtggcggagggagcggcggtggaggaagcggaggcggaggttccGATTGGCTCACACAATCCCCTGCAATCCTGAGTGCATCTCCAGGCGAGAAAGTAACTATGACTTGCAGAGCTATAAGCTCTGTGTCCTACATGCACTGGTATCAGCAGAAGCCAGGTTCTTCCCCGAAGCCGTGGATATATGCTACAAGCAATTTGGCATCCGGTGTTCCCGCCCGGTTTAGTGGCTCCGGTTCTGGGACAAGTTACTCCCTCACGATCAGCAGGGTTGAAGCCGAGGACGCTGCCACTTACTATTGCCAACAGTGGTCAAGTAACCCCAGGACTTTCGGGGGAGGAACTAAACTTGAAATCAA AtctagaCD5-34L2H-3053 CAR (SEQ ID NO: 12)Ggatcc GAT TGG CTC ACA CAA TCC CCT GCA ATC CTG AGT GCA TCT CCA GGCGAG AAA GTA ACT ATG ACT TGC AGA GCT ATA AGC TCT GTG TCC TAC ATGCAC TGG TAT CAG CAG AAG CCA GGT TCT TCC CCG AAG CCG TGG ATA TATGCT ACA AGC AAT TTG GCA TCC GGT GTT CCC GCC CGG TTT AGT GGC TCCGGT TCT GGG ACA AGT TAC TCC CTC ACG ATC AGC AGG GTT GAA GCC GAGGAC GCT GCC ACT TAC TAT TGC CAA CAG TGG TCA AGT AAC CCC AGG ACTTTC GGG GGA GGA ACT AAA CTT GAA ATC AAAGgtggcggagggagcggcggtggaggaagcggaggcggaggttcc GAG GTT AAA CTC GTG GAG AGCGGT GCC GAA CTC GTC CGA AGT GGT GCT TCC GTT AAA CTC AGT TGT GCCGCG TCA GGA TTT AAC ATA AAA GAT TAC TAC ATT CAC TGG GTC AAA CAGCGC CCG GAG CAG GGG CTT GAA TGG ATC GGG TGG ATT GAT CCT GAA AACGGG CGC ACC GAA TAT GCT CCC AAG TTC CAG GGC AAA GCT ACT ATG ACCGCT GAC ACC TCT AGT AAC ACT GCC TAC CTG CAG TTG AGC TCT CTT ACGTCT GAG GAT ACC GCT GTG TAC TAC TGT AAT AAC GGA AAT TAT GTA CGACAC TAT TAC TTC GAC TAC TGG GGG CAG GGC ACT ACT GTG ACT GTA TCTAGC tCTAGA CD5-17H2L-3054 CAR (SEQ ID NO: 13)ggatccGAGGTCCAACTCGTAGAATCAGGTCCCGGATTGGTGCAACCATCCCAGAGCCTCTCTATTACATGCACGGTCTCTGGATTTAGTCTGACCAATTACGATGTGCATTGGGTGCGCCAGTCTCCCGGCAAGGGGTTGGAATGGCTTGGCGTTATATGGAACTACGGAAATACAGACTATAACGCCGCGTTTATCTCTCGGCTGAGTATACGGAAAGACAGTAGTAAATCCCAGGTCTTTTTTACGATGTCATCCCTGCAAACGCCAGATACCGCAATATATTACTGCGCCAGGAACCACGGTGATGGTTATTATAATTGGTACTTCGATGTGTGGGGTACTGGCACTACAGTCACAGTATCTTCAggtggcggagggagcggcggtggaggaagcggaggcggaggttccAACATTGTACTGACGCAAAGCCCCTCATCTTTGTCTGAGTCACTCGGCGGCAAAGTAACCATCACATGCAAGGCCAGTCAAGACATCAATAAATATATTGCTTGGTATCAGTATAAACCCGGCAAGGGGCCGCGACTGCTGATTCACTACACGAGTACCTTGCAACCGGGCATTCCGAGCCGATTTAGTGGCAGTGGCTCAGGTCGCGATTACTCATTCTCAATAAGTAATCTCGAACCGGAAGACATAGCTACTTATTATTGCTTGCAGTACGATAATTTGTGGACCTTCGGGGGTGGTACAAAGTTGGAAA TAAAGtctagaCD8 Transmembrane domain nucleic acid sequence (SEQ ID NO: 14):atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatcaccctttact gcCD8 Transmembrane domain amino acid sequence (SEQ ID NO: 15):IYTWAPLAGTCGVLLLSLVITLYCCD8 hinge domain nucleic acid sequence (SEQ ID NO: 16):accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctgtccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctggacttcgcct gtgat CD8 hinge domain amino acid sequence (SEQ ID NO: 17):TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD4-1BB nucleic acid sequence (SEQ ID NO: 18)aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaaactactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt gaactgCD3-zeta nucleic acid sequence (SEQ ID NO: 19)agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctctataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggccgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaatgaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgccggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacctacgacgccc ttcacatgca ggccctgccc cctcgc4-1BB amino acid sequence (SEQ ID NO: 20):KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELCD3-zeta amino acid sequence (SEQ ID NO: 21):RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPRCD2-MEDI507H2L-3028 CAR amino acid sequence (SEQ ID NO: 25)MALPVTALLLPLALLLHAARPGSQVQLVQSGAEVQRPGASVKVSCKASGYIFTEYYMYWVRQAPGQGLELVGRIDPEDGSIDYVEKFKKKVTLTADTSSSTAYMELSSLTSDDTAVYYCARGKFNYRFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQSPPSLLVTLGQPASISCRSSQSLLHSSGNTYLNWLLQRPGQSPQPLIYLVSKLESGVPDRFSGSGSGTDFTLKISGVEAEDVGVYYCMQFTHYPYTFGQGTKLEIKSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRCD2-MEDI507L2H-3043 CAR amino acid sequence (SEQ ID NO: 26)MALPVTALLLPLALLLHAARPGSDVVMTQSPPSLLVTLGQPASISCRSSQSLLHSSGNTYLNWLLQRPGQSPQPLIYLVSKLESGVPDRFSGSGSGTDFTLKISGVEAEDVGVYYCMQFTHYPYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVQRPGASVKVSCKASGYIFTEYYMYWVRQAPGQGLELVGRIDPEDGSIDYVEKFKKKVTLTADTSSSTAYMELSSLTSDDTAVYYCARGKFNYRFAYWGQGTLVTVSSSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRCD2-MEDI507H2L-3028 scFv amino acid sequence (SEQ ID NO: 27)GSQVQLVQSGAEVQRPGASVKVSCKASGYIFTEYYMYWVRQAPGQGLELVGRIDPEDGSIDYVEKFKKKVTLTADTSSSTAYMELSSLTSDDTAVYYCARGKFNYRFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQSPPSLLVTLGQPASISCRSSQSLLHSSGNTYLNWLLQRPGQSPQPLIYLVSKLESGVPDRFSGSGSGTDFTLKISGVEAEDVGVYYCMQFTHYPYTFGQGTKLEIKSG CD2-MEDI507L2H-3043 scFv amino acid sequence(SEQ ID NO: 28)GSDVVMTQSPPSLLVTLGQPASISCRSSQSLLHSSGNTYLNWLLQRPGQSPQPLIYLVSKLESGVPDRFSGSGSGTDFTLKISGVEAEDVGVYYCMQFTHYPYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVQRPGASVKVSCKASGYIFTEYYMYWVRQAPGQGLELVGRIDPEDGSIDYVEKFKKKVTLTADTSSSTAYMELSSLTSDDTAVYYCARGKFNYRFAYWGQGTLVTVSSSG CD2-MEDI507 VH amino acid sequence(SEQ ID NO: 29) QVQLVQSGAEVQRPGASVKVSCKASGYIFTEYYMYWVRQAPGQGLELVGRIDPEDGSIDYVEKFKKKVTLTADTSSSTAYMELSSLTSDDTAVYYCARGKFNYRFAYWGQG TLVTVSSCD2-MEDI507 VL amino acid sequence (SEQ ID NO: 30)DVVMTQSPPSLLVTLGQPASISCRSSQSLLHSSGNTYLNWLLQRPGQSPQPLIYLVSKLESGVPDRFSGSGSGTDFTLKISGVEAEDVGVYYCMQFTHYPYTFGQGTKLEIK CD2-MEDI507 HCDR1(SEQ ID NO: 31) EYYMY CD2-MEDI507 HCDR2 (SEQ ID NO: 32)RIDPEDGSIDYVEKFKK CD2-MEDI507 HCDR3 (SEQ ID NO: 33) GKFNYRFAYCD2-MEDI507 LCDR1 (SEQ ID NO: 34) RSSPSLLHSSGNTYLN CD2-MEDI507 LCDR2(SEQ ID NO: 35) LVSKLES CD2-MEDI507 LCDR3 (SEQ ID NO: 36) MPFTHYPYTCD2-OKT11H2L-3029 CAR amino acid sequence (SEQ ID NO: 37)MALPVTALLLPLALLLHAARPGSPVPLPPPGAELVRPGTSVKLSCKASGYTFTSYWMHWIKPRPEPGLEWIGRIDPYDSETHYNEKFKDKAILSVDKSSSTAYIPLSSLTSDDSAVYYCSRRDAKYDGYALDYWGPGTTLTVSSGGGGSGGGGSGGGGSDIVMTQAAPSVPVTPGESVSISCRSSKTLLHSNGNTYLYWFLPRPGPSPPVLIYRMSNLASGVPNRFSGSGSETTFTLRISRVEAEDVGIYYCMPHLEYPYTFGGGTKLEIKSGTTTPAPRPPTPAPTIASPPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKPPFMRPVPTTPEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKPGPNPLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPPEGLYNELPKDKMAEAYSEIGMKGERRRGKGHDGLYPGLSTATKDTYDALHMPALPPRCD2-PKT11L2H-3030 CAR amino acid sequence (SEQ ID NO: 38)MALPVTALLLPLALLLHAARPGSDIVMTPAAPSVPVTPGESVSISCRSSKTLLHSNGNTYLYWFLPRPGPSPPVLIYRMSNLASGVPNRFSGSGSETTFTLRISRVEAEDVGIYYCMPHLEYPYTFGGGTKLEIKGGGGSGGGGSGGGGSPVPLPPPGAELVRPGTSVKLSCKASGYTFTSYWMHWIKPRPEPGLEWIGRIDPYDSETHYNEKFKDKAILSVDKSSSTAYIPLSSLTSDDSAVYYCSRRDAKYDGYALDYWGPGTTLTVSSSGTTTPAPRPPTPAPTIASPPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKPPFMRPVPTTPEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKPGPNPLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPPEGLYNELPKDKMAEAYSEIGMKGERRRGKGHDGLYPGLSTATKDTYDALHMPALPPRCD2-PKT11H2L-3029 scFv amino acid sequence (SEQ ID NO: 39)GSPVPLPPPGAELVRPGTSVKLSCKASGYTFTSYWMHWIKPRPEPGLEWIGRIDPYDSETHYNEKFKDKAILSVDKSSSTAYIPLSSLTSDDSAVYYCSRRDAKYDGYALDYWGPGTTLTVSSGGGGSGGGGSGGGGSDIVMTPAAPSVPVTPGESVSISCRSSKTLLHSNGNTYLYWFLPRPGPSPPVLIYRMSNLASGVPNRFSGSGSETTFTLRISRVEAEDVGIYYCMPHLEYPYTFGGGTKLEIKSG CD2-OKT11L2H-3030 scFv amino acid sequence(SEQ ID NO: 40)GSDIVMTPAAPSVPVTPGESVSISCRSSKTLLHSNGNTYLYWFLPRPGPSPPVLIYRMSNLASGVPNRFSGSGSETTFTLRISRVEAEDVGIYYCMPHLEYPYTFGGGTKLEIKGGGGSGGGGSGGGGSPVPLPPPGAELVRPGTSVKLSCKASGYTFTSYWMHWIKPRPEQGLEWIGRIDPYDSETHYNEKFKDKAILSVDKSSSTAYIQLSSLTSDDSAVYYCSRRDAKYDGYALDYWGQGTTLTVSSSG CD2-OKT11 VH amino acid sequence (SEQ ID NO: 41)QVQLQQPGAELVRPGTSVKLSCKASGYTFTSYWMHWIKQRPEQGLEWIGRIDPYDSETHYNEKFKDKAILSVDKSSSTAYIQLSSLTSDDSAVYYCSRRDAKYDGYALDYWG QGTTLTVSSCD2-OKT11 VL amino acid sequence (SEQ ID NO: 42)DIVMTQAAPSVPVTPGESVSISCRSSKTLLHSNGNTYLYWFLQRPGQSPQVLIYRMSNLASGVPNRFSGSGSETTFTLRISRVEAEDVGIYYCMQHLEYPYTFGGGTKLEIK CD2-OKT11 HCDR1(SEQ ID NO: 43) SYWMH CD2-OKT11 HCDR2 (SEQ ID NO: 44) RIDPYDSETHYNEKFKDCD2-OKT11 HCDR3 (SEQ ID NO: 45) RDAKYDGYALDY CD2-OKT11 LCDR1(SEQ ID NO: 46) RSSKTLLHSNGNTYLY CD2-OKT11 LCDR2 (SEQ ID NO: 47) RMSNLASCD2-OKT11 LCDR3 (SEQ ID NO: 48) MPHLEYPYTCD2-T11-2-H2L-3031 CAR amino acid sequence (SEQ ID NO: 49)MALPVTALLLPLALLLHAARPGSPVPLPPPGAELVRPGASVKLSCKASGYTFTTFWMNWVKPRPGPGLEWIGMIDPSDSEAHYNPMFKDKATLTVDKSSSTAYMPLSSLTSEDSAVYYCARGRGYDDGDAMDYWGPGTSVTVSSGGGGSGGGGSGGGGSDIVMTPSPASLAVSLGPRATISYRASKSVSTSGYSYMHWNPPKPGPPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCMPFTHYPYTFGGGTKLEIKSGTTTPAPRPPTPAPTIASPPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKPPFMRPVPTTPEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKPGPNPLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPPEGLYNELPKDKMAEAYSEIGMKGERRRGKGHDGLYPGLSTATKDTYDALHMPALPPRCD2-T11-2-H2L-3031 scFv amino acid sequence (SEQ ID NO: 50)GSPVPLPPPGAELVRPGASVKLSCKASGYTFTTFWMNWVKPRPGPGLEWIGMIDPSDSEAHYNPMFKDKATLTVDKSSSTAYMPLSSLTSEDSAVYYCARGRGYDDGDAMDYWGPGTSVTVSSGGGGSGGGGSGGGGSDIVMTPSPASLAVSLGPRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCMQFTHYPYTFGGGTKLEIKSG CD2-T11-2-H2L-3031 VH amino acid sequence(SEQ ID NO: 51) QVQLQQPGAELVRPGASVKLSCKASGYTFTTFWMNWVKQRPGQGLEWIGMIDPSDSEAHYNPMFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGRGYDDGDAMD YWGQGTSVTVSSCD2-T11-2-H2L-3031 VL amino acid sequence (SEQ ID NO: 52)DIVMTPSPASLAVSLGPRATISYRASKSVSTSGYSYMHWNPPKPGPPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCMPFTHYPYTFGGGTKLEIK CD2-T11-2 HCDR1(SEQ ID NO: 53) TFWMN CD2-T11-2 HCDR2 (SEQ ID NO: 54) MIDPSDSEAHYNQMFKDCD2-T11-2 HCDR3 (SEQ ID NO: 55) GRGYDDGDAMDY CD2-T11-2 LCDR1(SEQ ID NO: 56) RASKSVSTSGYSYMH CD2-T11-2 LCDR2 (SEQ ID NO: 57) LVSNLESCD2-T11-2 LCDR3 (SEQ ID NO: 58) MQFTHYPYTCD2-TS2-18.1.1-H2L-3032 CAR amino acid sequence (SEQ ID NO: 59)MALPVTALLLPLALLLHAARPGSEVPLEESGGGLVMPGGSLKLSCAASGFAFSSYDMSWVRPTPEKRLEWVAYISGGGFTYYPDTVKGRFTLSRDNAKNTLYLPMSSLKSEDTAMYYCARPGANWELVYWGPGTTLTVSSGGGGSGGGGSGGGGSDIVMTPSPATLSVTPGDRVFLSCRASPSISDFLHWYPPKSHESPRLLIKYASPSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYLCPNGHNFPPTFGGGTKLEIKSGTTTPAPRPPTPAPTIASPPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKPPFMRPVPTTPEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKPGPNPLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPPEGLYNELPKDKMAEAYSEIGMKGERRRGKGHDGLYPGLSTATKDTYDALHMPALPPRCD2-TS2-18.1.1-L2H-3033 CAR amino acid sequence (SEQ ID NO: 60)MALPVTALLLPLALLLHAARPGSDIVMTPSPATLSVTPGDRVFLSCRASPSISDFLHWYPPKSHESPRLLIKYASPSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYLCPNGHNFPPTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLEESGGGLVMPGGSLKLSCAASGFAFSSYDMSWVRPTPEKRLEWVAYISGGGFTYYPDTVKGRFTLSRDNAKNTLYLPMSSLKSEDTAMYYCARQGANWELVYWGQGTTLTVSSSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRCD2-TS2-18.1.1-H2L-3032 scFv amino acid sequence (SEQ ID NO: 61)GSEVQLEESGGGLVMPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVAYISGGGFTYYPDTVKGRFTLSRDNAKNTLYLQMSSLKSEDTAMYYCARQGANWELVYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQSPATLSVTPGDRVFLSCRASQSISDFLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYLCQNGHNFPPTFGGGTKLEIKSG CD2-TS2-18.1.1-L2H-3033 scFv amino acid sequence(SEQ ID NO: 62)GSDIVMTQSPATLSVTPGDRVFLSCRASQSISDFLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYLCQNGHNFPPTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLEESGGGLVMPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVAYISGGGFTYYPDTVKGRFTLSRDNAKNTLYLQMSSLKSEDTAMYYCARQGANWE LVYWGQGTTLTVSSSGCD2-TS2-18.1.1 VH amino acid sequence (SEQ ID NO: 63)EVQLEESGGGLVMPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVAYISGGGFTYYPDTVKGRFTLSRDNAKNTLYLQMSSLKSEDTAMYYCARQGANWELVYWGQG TTLTVSSCD2-TS2-18.1.1 VL amino acid sequence (SEQ ID NO: 64)DIVMTQSPATLSVTPGDRVFLSCRASQSISDFLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYLCQNGHNFPPTFGGGTKLEIK CD2-TS2-18.1.1 HCDR1(SEQ ID NO: 65) SYDMS CD2-TS2-18.1.1 HCDR2 (SEQ ID NO: 66)YISGGGFTYYPDTVKG CD2-TS2-18.1.1 HCDR3 (SEQ ID NO: 67) PGANWELVYCD2-TS2-18.1.1 LCDR1 (SEQ ID NO: 68) RASQSISDFLH CD2-TS2-18.1.1 LCDR2(SEQ ID NO: 69) YASQSIS CD2-TS2-18.1.1 LCDR3 (SEQ ID NO: 70) QNGHNFPPTCD5-17L2H-3045 CAR amino acid sequence (SEQ ID NO: 71)MALPVTALLLPLALLLHAARPGSNIVLTQSPSSLSESLGGKVTITCKASQDINKYIAWYPYKPGKGPRLLIHYTSTLPPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLPYDNLWTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGPGLVQPSQSLSITCTVSGFSLTNYDVHWVRPSPGKGLEWLGVIWNYGNTDYNAAFISRLSIRKDSSKSPVFFTMSSLPTPDTAIYYCARNHGDGYYNWYFDVWGTGTTVTVSSSRTTTPAPRPPTPAPTIASPPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCHMKRGRKKLLYIFKPPFMRPVPTTPEEDGCSCRFPEEEEGGCELTSRVKFSRSADAPAYPPGPNPLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPPEGLYNELPKDKMAEAYSEIGMKGERRRGKGHDGLYPGLSTATKDTYDALHMPALPPRCD5-17H2L-3054 CAR amino acid sequence (SEQ ID NO: 72)MALPVTALLLPLALLLHAARPGSEVPLVESGPGLVPPSPSLSITCTVSGFSLTNYDVHWVRPSPGKGLEWLGVIWNYGNTDYNAAFISRLSIRKDSSKSPVFFTMSSLPTPDTAIYYCARNHGDGYYNWYFDVWGTGTTVTVSSGGGGSGGGGSGGGGSNIVLTQSPSSLSESLGGKVTITCKASQDINKYIAWYQYKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLPYDNLWTFGGGTKLEIKSRTTTPAPRPPTPAPTIASPPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCHMKRGRKKLLYIFKPPFMRPVPTTPEEDGCSCRFPEEEEGGCELTSRVKFSRSADAPAYPPGPNPLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPPEGLYNELPKDKMAEAYSEIGMKGERRRGKGHDGLYPGLSTATKDTYDALHMPALPPRCD5-17L2H-3045 scFv amino acid sequence (SEQ ID NO: 73)NIVLTPSPSSLSESLGGKVTITCKASPDINKYIAWYPYKPGKGPRLLIHYTSTLPPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLPYDNLWTFGGGTKLEIKGGGGSGGGGSGGGGSEVPLVESGPGLVPPSPSLSITCTVSGFSLTNYDVHWVRPSPGKGLEWLGVIWNYGNTDYNAAFISRLSIRKDSSKSPVFFTMSSLPTPDTAIYYCARNHGDGYYNWY FDVWGTGTTVTVSSCD5-17H2L-3054 scFv amino acid sequence (SEQ ID NO: 74)EVPLVESGPGLVPPSPSLSITCTVSGFSLTNYDVHWVRPSPGKGLEWLGVIWNYGNTDYNAAFISRLSIRKDSSKSPVFFTMSSLPTPDTAIYYCARNHGDGYYNWYFDVWGTGTTVTVSSGGGGSGGGGSGGGGSNIVLTPSPSSLSESLGGKVTITCKASPDINKYIAWYPYKPGKGPRLLIHYTSTLPPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLPYDNLWTFGGGTKLEIK CD5-17 VH amino acid sequence (SEQ ID NO: 75)EVPLVESGPGLVPPSPSLSITCTVSGFSLTNYDVHWVRPSPGKGLEWLGVIWNYGNTDYNAAFISRLSIRKDSSKSPVFFTMSSLPTPDTAIYYCARNHGDGYYNWYFDVWG TGTTVTVSSCD5-17 VL amino acid sequence (SEQ ID NO: 76)NIVLTPSPSSLSESLGGKVTITCKASPDINKYIAWYPYKPGKGPRLLIHYTSTLPPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLPYDNLWTFGGGTKLEIKCD5-9H2L-3048 CAR amino acid sequence (SEQ ID NO: 77)MALPVTALLLPLALLLHAARPGSQVQLKESGPELEKPGASVKISCKASGYSFTAYSMNWVKQNNGMSLEWIGSIDPYYGDTKYAQKFKGKATLTVDKASSTAYLQLKSLTSEDSAVYYCARRMITTGDWYFDVWGTGTTVTVSSGGGGSGGGGSGGGGSHIVLTQSPSSLSASLGDRVTISCRASQDISTYLNWYQQKPDGTVKLLIFYTSRLHAGVPSRFSGSGSGTHHSLTISNLEQEDIATYFCQQGNSLPFTFGSGTKLEIKSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRCD5-9L2H-3049 CAR amino acid sequence (SEQ ID NO: 78)MALPVTALLLPLALLLHAARPGSHIVLTQSPSSLSASLGDRVTISCRASQDISTYLNWYPPKPDGTVKLLIFYTSRLHAGVPSRFSGSGSGTHHSLTISNLEPEDIATYFCPPGNSLPFTFGSGTKLEIKGGGGSGGGGSGGGGSPVPLKESGPELEKPGASVKISCKASGYSFTAYSMNWVKPNNGMSLEWIGSIDPYYGDTKYAPKFKGKATLTVDKASSTAYLPLKSLTSEDSAVYYCARRMITTGDWYFDVWGTGTTVTVSSSGTTTPAPRPPTPAPTIASPPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKPPFMRPVPTTPEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKPGPNPLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPPEGLYNELPKDKMAEAYSEIGMKGERRRGKGHDGLYPGLSTATKDTYDALHMPALPPRCD5-9H2L-3048 scFv amino acid sequence (SEQ ID NO: 79)GSPVPLKESGPELEKPGASVKISCKASGYSFTAYSMNWVKPNNGMSLEWIGSIDPYYGDTKYAPKFKGKATLTVDKASSTAYLPLKSLTSEDSAVYYCARRMITTGDWYFDVWGTGTTVTVSSGGGGSGGGGSGGGGSHIVLTPSPSSLSASLGDRVTISCRASPDISTYLNWYPPKPDGTVKLLIFYTSRLHAGVPSRFSGSGSGTHHSLTISNLEPEDIATYFCPPGNSLPFTFGSGTKLEIKSG CD5-9L2H-3049 scFv amino acid sequence(SEQ ID NO: 80)HIVLTPSPSSLSASLGDRVTISCRASPDISTYLNWYPPKPDGTVKLLIFYTSRLHAGVPSRFSGSGSGTHHSLTISNLEPEDIATYFCPPGNSLPFTFGSGTKLEIKGGGGSGGGGSGGGGSPVPLKESGPELEKPGASVKISCKASGYSFTAYSMNWVKPNNGMSLEWIGSIDPYYGDTKYAPKFKGKATLTVDKASSTAYLPLKSLTSEDSAVYYCARRMITTGDW YFDVWGTGTTVTVSSCD5-9 VH amino acid sequence (SEQ ID NO: 81)PVPLKESGPELEKPGASVKISCKASGYSFTAYSMNWVKPNNGMSLEWIGSIDPYYGDTKYAPKFKGKATLTVDKASSTAYLPLKSLTSEDSAVYYCARRMITTGDWYFDVW GTGTTVTVSSCD5-9 VL amino acid sequence (SEQ ID NO: 82)HIVLTPSPSSLSASLGDRVTISCRASPDISTYLNWYPPKPDGTVKLLIFYTSRLHAGVPSRFSGSGSGTHHSLTISNLEPEDIATYFCPPGNSLPFTFGSGTKLEIK CD5-9 HCDR1(SEQ ID NO: 83) AYSMN CD5-9 HCDR2 (SEQ ID NO: 84) SIDPYYGDTKYAQKFKGCD5-9HCDR3 (SEQ ID NO: 85) RMITTGDWYFDV CD5-9 LCDR1 (SEQ ID NO: 86)RASPDISTYLN CD5-9 LCDR2 (SEQ ID NO: 87) YTSRLHA CD5-9 LCDR3(SEQ ID NO: 88) PPGNSLPFT CD5-34H2L-3052 CAR amino acid sequence(SEQ ID NO: 89)MALPVTALLLPLALLLHAARPGSEVKLVESGAELVRSGASVKLSCAASGFNIKDYYIHWVKPRPEPGLEWIGWIDPENGRTEYAPKFPGKATMTADTSSNTAYLPLSSLTSEDTAVYYCNNGNYVRHYYFDYWGPGTTVTVSSGGGGSGGGGSGGGGSDWLTPSPAILSASPGEKVTMTCRAISSVSYMHWYPPKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCPPWSSNPRTFGGGTKLEIKSRTTTPAPRPPTPAPTIASPPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCHMKRGRKKLLYIFKPPFMRPVPTTPEEDGCSCRFPEEEEGGCELTSRVKFSRSADAPAYPPGPNPLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPPEGLYNELPKDKMAEAYSEIGMKGERRRGKGHDGLYPGLSTATKDTYDALHMPALPPRCD5-34L2H-3053 CAR amino acid sequence (SEQ ID NO: 90)MALPVTALLLPLALLLHAARPGSDWLTPSPAILSASPGEKVTMTCRAISSVSYMHWYPPKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCPPWSSNPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVKLVESGAELVRSGASVKLSCAASGFNIKDYYIHWVKPRPEPGLEWIGWIDPENGRTEYAPKFPGKATMTADTSSNTAYLPLSSLTSEDTAVYYCNNGNYVRHYYFDYWGQGTTVTVSSSRTTTPAPRPPTPAPTIASPPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCHMKRGRKKLLYIFKPPFMRPVPTTPEEDGCSCRFPEEEEGGCELTSRVKFSRSADAPAYPPGPNPLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPPEGLYNELPKDKMAEAYSEIGMKGERRRGKGHDGLYPGLSTATKDTYDALHMPALPPRCD5-34H2L-3052 scFv amino acid sequence (SEQ ID NO: 91)EVKLVESGAELVRSGASVKLSCAASGFNIKDYYIHWVKPRPEPGLEWIGWIDPENGRTEYAPKFPGKATMTADTSSNTAYLPLSSLTSEDTAVYYCNNGNYVRHYYFDYWGPGTTVTVSSGGGGSGGGGSGGGGSDWLTPSPAILSASPGEKVTMTCRAISSVSYMHWYPPKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCPPWSSNPRTFGGGTKLEIK CD5-34L2H-3053 scFv amino acid sequence(SEQ ID NO: 92)DWLTQSPAILSASPGEKVTMTCRAISSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVKLVESGAELVRSGASVKLSCAASGFNIKDYYIHWVKQRPEQGLEWIGWIDPENGRTEYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNNGNYVRHYY FDYWGQGTTVTVSSCD5-34 VH amino acid sequence (SEQ ID NO: 93)EVKLVESGAELVRSGASVKLSCAASGFNIKDYYIHWVKQRPEQGLEWIGWIDPENGRTEYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNNGNYVRHYYFDYWG QGTTVTVSSCD5-34 VL amino acid sequence (SEQ ID NO: 94)DWLTQSPAILSASPGEKVTMTCRAISSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNPRTFGGGTKLEIKSR CD5-34 HCDR1(SEQ ID NO: 95) DYYIH CD5-34 HCDR2 (SEQ ID NO: 96) WIDPENGRTEYAPKFPGCD5-34 HCDR3 (SEQ ID NO: 97) GNYVRHYYFDY CD5-34 LCDR1 (SEQ ID NO: 98)RAISSVSYMH CD5-34 LCDR2 (SEQ ID NO: 99) ATSNLAS CD5-34 LCDR3(SEQ ID NO: 100) QQWSSNPRTCRISPR/Cas

Certain embodiments of the invention include cells that have beenmodified by a CRISPR/Cas system. CRISPR/Cas systems include, but are notlimited to, the CRISPR/Cas9 system and the CRISPR/Cpf1 system. Incertain embodiments, the invention includes cells that have beenmodified using the CRISPR/Cas9 system. In certain embodiments, themodifications include knocking-out or mutating an endogenous gene, e.g.CD2; CD5, or CD7.

The CRISPR/Cas9 system is a facile and efficient system for inducingtargeted genetic alterations. Target recognition by the Cas9 proteinrequires a ‘seed’ sequence within the guide RNA (gRNA or sgRNA) and aconserved tri-nucleotide containing protospacer adjacent motif (PAM)sequence upstream of the gRNA-binding region. The CRISPR/Cas9 system canthereby be engineered to cleave virtually any DNA sequence byredesigning the gRNA for use in cell lines (such as 293T cells), primarycells, and CAR T cells. The CRISPR/Cas system can simultaneously targetmultiple genomic loci by co-expressing a single Cas9 protein with two ormore gRNAs, making this system uniquely suited for multiple gene editingor synergistic activation of target genes.

One example of a CRISPR/Cas system used to inhibit gene expression,CRISPRi, is described in U.S. Publication No. US2014/0068797, which isincorporated herein by reference in its entirety. CRISPRi inducespermanent gene disruption that utilizes the RNA-guided Cas9 endonucleaseto introduce DNA double stranded breaks which trigger error-prone repairpathways to result in frame shift mutations. A catalytically dead Cas9lacks endonuclease activity. When coexpressed with a guide RNA, a DNArecognition complex is generated that specifically interferes withtranscriptional elongation, RNA polymerase binding, or transcriptionfactor binding. This CRISPRi system efficiently represses expression oftargeted genes.

CRISPR/Cas gene disruption occurs when a guide nucleic acid sequencespecific for a target gene and a Cas endonuclease are introduced into acell and form a complex that enables the Cas endonuclease to introduce adouble strand break at the target gene. In certain embodiments, theCRISPR system comprises an expression vector, such as, but not limitedto, an pAd5F35-CRISPR vector. In other embodiments, the Cas expressionvector induces expression of Cas9 endonuclease. Other endonucleases mayalso be used, including but not limited to Cpf1, T7, Cas3, Cas8a, Cas8b,Cas10d, Cse1, Csy1, Csn2, Cas4, Cas10, Csm2, Cmr5, Fok1, other nucleasesknown in the art, and any combination thereof.

In certain embodiments, inducing the Cas expression vector comprisesexposing the cell to an agent that activates an inducible promoter inthe Cas expression vector. In such embodiments, the Cas expressionvector includes an inducible promoter, such as one that is inducible byexposure to an antibiotic (e.g., by tetracycline or a derivative oftetracycline, for example doxycycline). However, it should beappreciated that other inducible promoters can be used. The inducingagent can be a selective condition (e.g., exposure to an agent, forexample an antibiotic) that results in induction of the induciblepromoter. This results in expression of the Cas expression vector.

The guide nucleic acid sequence is specific for a gene and targets thatgene for Cas endonuclease-induced double strand breaks. The sequence ofthe guide nucleic acid sequence may be within a loci of the gene. In oneembodiment, the guide nucleic acid sequence is at least 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40 or more nucleotides in length.

The guide nucleic acid sequence may be specific for any gene, e.g. CD2,CD5, CD7. The guide nucleic acid sequence includes a RNA sequence, a DNAsequence, a combination thereof (a RNA-DNA combination sequence), or asequence with synthetic nucleotides. The guide nucleic acid sequence canbe a single molecule or a double molecule. In one embodiment, the guidenucleic acid sequence comprises a single guide RNA.

In the context of formation of a CRISPR complex, “target sequence”refers to a sequence to which a guide sequence is designed to have somecomplementarity, where hybridization between a target sequence and aguide sequence promotes the formation of a CRISPR complex. Fullcomplementarity is not necessarily required, provided there issufficient complementarity to cause hybridization and promote formationof a CRISPR complex. A target sequence may comprise any polynucleotide,such as DNA or RNA polynucleotides. In certain embodiments, a targetsequence is located in the nucleus or cytoplasm of a cell. In otherembodiments, the target sequence may be within an organelle of aeukaryotic cell, for example, mitochondrion or nucleus. Typically, inthe context of an endogenous CRISPR system, formation of a CRISPRcomplex (comprising a guide sequence hybridized to a target sequence andcomplexed with one or more Cas proteins) results in cleavage of one orboth strands in or near (e.g., within about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 50 or more base pairs) the target sequence. As with the targetsequence, it is believed that complete complementarity is not needed,provided this is sufficient to be functional. In certain embodiments,the tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% ofsequence complementarity along the length of the tracr mate sequencewhen optimally aligned.

In other embodiments, one or more vectors driving expression of one ormore elements of a CRISPR system are introduced into a host cell, suchthat expression of the elements of the CRISPR system direct formation ofa CRISPR complex at one or more target sites. For example, a Cas enzyme,a guide sequence linked to a tracr-mate sequence, and a tracr sequencecould each be operably linked to separate regulatory elements onseparate vectors. Alternatively, two or more of the elements expressedfrom the same or different regulatory elements may be combined in asingle vector, with one or more additional vectors providing anycomponents of the CRISPR system not included in the first vector. CRISPRsystem elements that are combined in a single vector may be arranged inany suitable orientation, such as one element located 5′ with respect to(“upstream” of) or 3′ with respect to (“downstream” of) a secondelement. The coding sequence of one element may be located on the sameor opposite strand of the coding sequence of a second element, andoriented in the same or opposite direction. In certain embodiments, asingle promoter drives expression of a transcript encoding a CRISPRenzyme and one or more of the guide sequence, tracr mate sequence(optionally operably linked to the guide sequence), and a tracr sequenceembedded within one or more intron sequences (e.g., each in a differentintron, two or more in at least one intron, or all in a single intron).

In certain embodiments, the CRISPR enzyme is part of a fusion proteincomprising one or more heterologous protein domains (e.g. about or morethan about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition tothe CRISPR enzyme). A CRISPR enzyme fusion protein may comprise anyadditional protein sequence, and optionally a linker sequence betweenany two domains. Examples of protein domains that may be fused to aCRISPR enzyme include, without limitation, epitope tags, reporter genesequences, and protein domains having one or more of the followingactivities: methylase activity, demethylase activity, transcriptionactivation activity, transcription repression activity, transcriptionrelease factor activity, histone modification activity, RNA cleavageactivity and nucleic acid binding activity. Additional domains that mayform part of a fusion protein comprising a CRISPR enzyme are describedin US20110059502, incorporated herein by reference. In certainembodiments, a tagged CRISPR enzyme is used to identify the location ofa target sequence.

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids in mammalian cells or target tissues. Suchmethods can be used to administer nucleic acids encoding components of aCRISPR system to cells in culture, or in a host organism. Non-viralvector delivery systems include DNA plasmids, RNA (e.g. a transcript ofa vector described herein), naked nucleic acid, and nucleic acidcomplexed with a delivery vehicle, such as a liposome. Another deliverymode for the CRISPR/Cas9 comprises a combination of RNA and purifiedCas9 protein in the form of a Cas9-guide RNA ribonucleoprotein (RNP)complex. (Lin et al., 2014, ELife 3:e04766). Viral vector deliverysystems include DNA and RNA viruses, which have either episomal orintegrated genomes after delivery to the cell (Anderson, 1992, Science256:808-813; and Yu et al., 1994, Gene Therapy 1:13-26).

In certain embodiments, the CRISPR/Cas is derived from a type IICRISPR/Cas system. In other embodiments, the CRISPR/Cas system isderived from a Cas9 protein. The Cas9 protein can be from Streptococcuspyogenes, Streptococcus thermophilus, or other species. In certainembodiments, Cas9 can include: spCas9, Cpf1, CasY, CasX, or saCas9.

In general, CRISPR/Cas proteins comprise at least one RNA recognitionand/or RNA binding domain. RNA recognition and/or RNA binding domainsinteract with the guiding RNA. CRISPR/Cas proteins can also comprisenuclease domains (i.e., DNase or RNase domains), DNA binding domains,helicase domains, RNAse domains, protein-protein interaction domains,dimerization domains, as well as other domains. The CRISPR/Cas proteinscan be modified to increase nucleic acid binding affinity and/orspecificity, alter an enzymatic activity, and/or change another propertyof the protein. In certain embodiments, the CRISPR/Cas-like protein ofthe fusion protein can be derived from a wild type Cas9 protein orfragment thereof. In other embodiments, the CRISPR/Cas can be derivedfrom modified Cas9 protein. For example, the amino acid sequence of theCas9 protein can be modified to alter one or more properties (e.g.,nuclease activity, affinity, stability, and so forth) of the protein.Alternatively, domains of the Cas9 protein not involved in RNA-guidedcleavage can be eliminated from the protein such that the modified Cas9protein is smaller than the wild type Cas9 protein. In general, a Cas9protein comprises at least two nuclease (i.e., DNase) domains. Forexample, a Cas9 protein can comprise a RuvC-like nuclease domain and aHNH-like nuclease domain. The RuvC and HNH domains work together to cutsingle strands to make a double-stranded break in DNA. (Jinek et al.,2012, Science, 337:816-821). In certain embodiments, the Cas9-derivedprotein can be modified to contain only one functional nuclease domain(either a RuvC-like or a HNH-like nuclease domain). For example, theCas9-derived protein can be modified such that one of the nucleasedomains is deleted or mutated such that it is no longer functional(i.e., the nuclease activity is absent). In some embodiments in whichone of the nuclease domains is inactive, the Cas9-derived protein isable to introduce a nick into a double-stranded nucleic acid (suchprotein is termed a “nickase”), but not cleave the double-stranded DNA.In any of the above-described embodiments, any or all of the nucleasedomains can be inactivated by one or more deletion mutations, insertionmutations, and/or substitution mutations using well-known methods, suchas site-directed mutagenesis, PCR-mediated mutagenesis, and total genesynthesis, as well as other methods known in the art.

In one non-limiting embodiment, a vector drives the expression of theCRISPR system. The art is replete with suitable vectors that are usefulin the present invention. The vectors to be used are suitable forreplication and, optionally, integration in eukaryotic cells. Typicalvectors contain transcription and translation terminators, initiationsequences, and promoters useful for regulation of the expression of thedesired nucleic acid sequence. The vectors of the present invention mayalso be used for nucleic acid standard gene delivery protocols. Methodsfor gene delivery are known in the art (U.S. Pat. Nos. 5,399,346,5,580,859 & 5,589,466, incorporated by reference herein in theirentireties).

Further, the vector may be provided to a cell in the form of a viralvector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al. (4^(th) Edition, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York,2012), and in other virology and molecular biology manuals. Viruses,which are useful as vectors include, but are not limited to,retroviruses, adenoviruses, adeno-associated viruses, herpes viruses,Sindbis virus, gammaretrovirus and lentiviruses. In general, a suitablevector contains an origin of replication functional in at least oneorganism, a promoter sequence, convenient restriction endonucleasesites, and one or more selectable markers (e.g., WO 01/96584; WO01/29058; and U.S. Pat. No. 6,326,193).

Introduction of Nucleic Acids

Methods of introducing nucleic acids into a cell include physical,biological and chemical methods. Physical methods for introducing apolynucleotide, such as RNA, into a host cell include calcium phosphateprecipitation, lipofection, particle bombardment, microinjection,electroporation, and the like. RNA can be introduced into target cellsusing commercially available methods which include electroporation(Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830(BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II(BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany). RNAcan also be introduced into cells using cationic liposome mediatedtransfection using lipofection, using polymer encapsulation, usingpeptide mediated transfection, or using biolistic particle deliverysystems such as “gene guns” (see, for example, Nishikawa, et al. HumGene Ther., 12(8):861-70 (2001).

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). However, compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentinvention, in order to confirm the presence of the nucleic acids in thehost cell, a variety of assays may be performed. Such assays include,for example, “molecular biological” assays well known to those of skillin the art, such as Southern and Northern blotting, RT-PCR and PCR;“biochemical” assays, such as detecting the presence or absence of aparticular peptide, e.g., by immunological means (ELISAs and Westernblots) or by assays described herein to identify agents falling withinthe scope of the invention.

Moreover, the nucleic acids may be introduced by any means, such astransducing the expanded T cells, transfecting the expanded T cells, andelectroporating the expanded T cells. One nucleic acid may be introducedby one method and another nucleic acid may be introduced into the T cellby a different method.

RNA

In one embodiment, the nucleic acids introduced into the T cell are RNA.In another embodiment, the RNA is mRNA that comprises in vitrotranscribed RNA or synthetic RNA. The RNA is produced by in vitrotranscription using a polymerase chain reaction (PCR)-generatedtemplate. DNA of interest from any source can be directly converted byPCR into a template for in vitro mRNA synthesis using appropriateprimers and RNA polymerase. The source of the DNA can be, for example,genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or anyother appropriate source of DNA. The desired template for in vitrotranscription is a chimeric membrane protein. By way of example, thetemplate encodes an antibody, a fragment of an antibody or a portion ofan antibody. By way of another example, the template comprises anextracellular domain comprising a single chain variable domain of anantibody, such as anti-CD3, and an intracellular domain of aco-stimulatory molecule. In one embodiment, the template for the RNAchimeric membrane protein encodes a chimeric membrane protein comprisingan extracellular domain comprising an antigen binding domain derivedfrom an antibody to a co-stimulatory molecule, and an intracellulardomain derived from a portion of an intracellular domain of CD28 and4-1BB.

PCR can be used to generate a template for in vitro transcription ofmRNA which is then introduced into cells. Methods for performing PCR arewell known in the art. Primers for use in PCR are designed to haveregions that are substantially complementary to regions of the DNA to beused as a template for the PCR. “Substantially complementary”, as usedherein, refers to sequences of nucleotides where a majority or all ofthe bases in the primer sequence are complementary, or one or more basesare non-complementary, or mismatched. Substantially complementarysequences are able to anneal or hybridize with the intended DNA targetunder annealing conditions used for PCR. The primers can be designed tobe substantially complementary to any portion of the DNA template. Forexample, the primers can be designed to amplify the portion of a genethat is normally transcribed in cells (the open reading frame),including 5′ and 3′ UTRs. The primers can also be designed to amplify aportion of a gene that encodes a particular domain of interest. In oneembodiment, the primers are designed to amplify the coding region of ahuman cDNA, including all or portions of the 5′ and 3′ UTRs. Primersuseful for PCR are generated by synthetic methods that are well known inthe art. “Forward primers” are primers that contain a region ofnucleotides that are substantially complementary to nucleotides on theDNA template that are upstream of the DNA sequence that is to beamplified. “Upstream” is used herein to refer to a location 5, to theDNA sequence to be amplified relative to the coding strand. “Reverseprimers” are primers that contain a region of nucleotides that aresubstantially complementary to a double-stranded DNA template that aredownstream of the DNA sequence that is to be amplified. “Downstream” isused herein to refer to a location 3′ to the DNA sequence to beamplified relative to the coding strand.

Chemical structures that have the ability to promote stability and/ortranslation efficiency of the RNA may also be used. The RNA preferablyhas 5′ and 3′ UTRs. In one embodiment, the 5′ UTR is between zero and3000 nucleotides in length. The length of 5′ and 3′ UTR sequences to beadded to the coding region can be altered by different methods,including, but not limited to, designing primers for PCR that anneal todifferent regions of the UTRs. Using this approach, one of ordinaryskill in the art can modify the 5′ and 3′ UTR lengths required toachieve optimal translation efficiency following transfection of thetranscribed RNA.

The 5′ and 3′ UTRs can be the naturally occurring, endogenous 5′ and 3′UTRs for the gene of interest. Alternatively, UTR sequences that are notendogenous to the gene of interest can be added by incorporating the UTRsequences into the forward and reverse primers or by any othermodifications of the template. The use of UTR sequences that are notendogenous to the gene of interest can be useful for modifying thestability and/or translation efficiency of the RNA. For example, it isknown that AU-rich elements in 3′ UTR sequences can decrease thestability of mRNA. Therefore, 3′ UTRs can be selected or designed toincrease the stability of the transcribed RNA based on properties ofUTRs that are well known in the art.

In one embodiment, the 5′ UTR can contain the Kozak sequence of theendogenous gene. Alternatively, when a 5′ UTR that is not endogenous tothe gene of interest is being added by PCR as described above, aconsensus Kozak sequence can be redesigned by adding the 5′ UTRsequence. Kozak sequences can increase the efficiency of translation ofsome RNA transcripts, but does not appear to be required for all RNAs toenable efficient translation. The requirement for Kozak sequences formany mRNAs is known in the art. In other embodiments the 5′ UTR can bederived from an RNA virus whose RNA genome is stable in cells. In otherembodiments various nucleotide analogues can be used in the 3′ or 5′ UTRto impede exonuclease degradation of the mRNA.

To enable synthesis of RNA from a DNA template without the need for genecloning, a promoter of transcription should be attached to the DNAtemplate upstream of the sequence to be transcribed. When a sequencethat functions as a promoter for an RNA polymerase is added to the 5′end of the forward primer, the RNA polymerase promoter becomesincorporated into the PCR product upstream of the open reading framethat is to be transcribed. In one embodiment, the promoter is a T7polymerase promoter, as described elsewhere herein. Other usefulpromoters include, but are not limited to, T3 and SP6 RNA polymerasepromoters. Consensus nucleotide sequences for T7, T3 and SP6 promotersare known in the art.

In one embodiment, the mRNA has both a cap on the 5′ end and a 3′poly(A) tail which determine ribosome binding, initiation of translationand stability mRNA in the cell. On a circular DNA template, forinstance, plasmid DNA, RNA polymerase produces a long concatamericproduct which is not suitable for expression in eukaryotic cells. Thetranscription of plasmid DNA linearized at the end of the 3′ UTR resultsin normal sized mRNA which is not effective in eukaryotic transfectioneven if it is polyadenylated after transcription.

On a linear DNA template, phage T7 RNA polymerase can extend the 3′ endof the transcript beyond the last base of the template (Schenborn andMierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva andBerzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

The conventional method of integration of polyA/T stretches into a DNAtemplate is molecular cloning. However polyA/T sequence integrated intoplasmid DNA can cause plasmid instability, which is why plasmid DNAtemplates obtained from bacterial cells are often highly contaminatedwith deletions and other aberrations. This makes cloning procedures notonly laborious and time consuming but often not reliable. That is why amethod which allows construction of DNA templates with polyA/T 3′stretch without cloning highly desirable.

The polyA/T segment of the transcriptional DNA template can be producedduring PCR by using a reverse primer containing a polyT tail, such as100T tail (size can be 50-5000 T), or after PCR by any other method,including, but not limited to, DNA ligation or in vitro recombination.Poly(A) tails also provide stability to RNAs and reduce theirdegradation. Generally, the length of a poly(A) tail positivelycorrelates with the stability of the transcribed RNA. In one embodiment,the poly(A) tail is between 100 and 5000 adenosines.

Poly(A) tails of RNAs can be further extended following in vitrotranscription with the use of a poly(A) polymerase, such as E. colipolyA polymerase (E-PAP). In one embodiment, increasing the length of apoly(A) tail from 100 nucleotides to between 300 and 400 nucleotidesresults in about a two-fold increase in the translation efficiency ofthe RNA. Additionally, the attachment of different chemical groups tothe 3′ end can increase mRNA stability. Such attachment can containmodified/artificial nucleotides, aptamers and other compounds. Forexample, ATP analogs can be incorporated into the poly(A) tail usingpoly(A) polymerase. ATP analogs can further increase the stability ofthe RNA.

5′ caps also provide stability to RNA molecules. In a preferredembodiment, RNAs produced by the methods disclosed herein include a 5′cap. The 5′ cap is provided using techniques known in the art anddescribed herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444(2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al.,Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

The RNAs produced by the methods disclosed herein can also contain aninternal ribosome entry site (IRES) sequence. The IRES sequence may beany viral, chromosomal or artificially designed sequence which initiatescap-independent ribosome binding to mRNA and facilitates the initiationof translation. Any solutes suitable for cell electroporation, which cancontain factors facilitating cellular permeability and viability such assugars, peptides, lipids, proteins, antioxidants, and surfactants can beincluded.

In some embodiments, the RNA is electroporated into the cells, such asin vitro transcribed RNA.

The disclosed methods can be applied to the modulation of T cellactivity in basic research and therapy, in the fields of cancer, stemcells, acute and chronic infections, and autoimmune diseases, includingthe assessment of the ability of the genetically modified T cell to killa target cancer cell.

The methods also provide the ability to control the level of expressionover a wide range by changing, for example, the promoter or the amountof input RNA, making it possible to individually regulate the expressionlevel. Furthermore, the PCR-based technique of mRNA production greatlyfacilitates the design of the mRNAs with different structures andcombination of their domains.

One advantage of RNA transfection methods of the invention is that RNAtransfection is essentially transient and a vector-free. A RNA transgenecan be delivered to a lymphocyte and expressed therein following a briefin vitro cell activation, as a minimal expressing cassette without theneed for any additional viral sequences. Under these conditions,integration of the transgene into the host cell genome is unlikely.Cloning of cells is not necessary because of the efficiency oftransfection of the RNA and its ability to uniformly modify the entirelymphocyte population.

Genetic modification of T cells with in vitro-transcribed RNA (IVT-RNA)makes use of two different strategies both of which have beensuccessively tested in various animal models. Cells are transfected within vitro-transcribed RNA by means of lipofection or electroporation. Itis desirable to stabilize IVT-RNA using various modifications in orderto achieve prolonged expression of transferred IVT-RNA.

Some IVT vectors are known in the literature which are utilized in astandardized manner as template for in vitro transcription and whichhave been genetically modified in such a way that stabilized RNAtranscripts are produced. Currently protocols used in the art are basedon a plasmid vector with the following structure: a 5′ RNA polymerasepromoter enabling RNA transcription, followed by a gene of interestwhich is flanked either 3′ and/or 5′ by untranslated regions (UTR), anda 3′ polyadenyl cassette containing 50-70 A nucleotides. Prior to invitro transcription, the circular plasmid is linearized downstream ofthe polyadenyl cassette by type II restriction enzymes (recognitionsequence corresponds to cleavage site). The polyadenyl cassette thuscorresponds to the later poly(A) sequence in the transcript. As a resultof this procedure, some nucleotides remain as part of the enzymecleavage site after linearization and extend or mask the poly(A)sequence at the 3′ end. It is not clear, whether this nonphysiologicaloverhang affects the amount of protein produced intracellularly fromsuch a construct.

RNA has several advantages over more traditional plasmid or viralapproaches. Gene expression from an RNA source does not requiretranscription and the protein product is produced rapidly after thetransfection. Further, since the RNA has to only gain access to thecytoplasm, rather than the nucleus, and therefore typical transfectionmethods result in an extremely high rate of transfection. In addition,plasmid based approaches require that the promoter driving theexpression of the gene of interest be active in the cells under study.

In another aspect, the RNA construct is delivered into the cells byelectroporation. See, e.g., the formulations and methodology ofelectroporation of nucleic acid constructs into mammalian cells astaught in US 2004/0014645, US 2005/0052630A1, US 2005/0070841A1, US2004/0059285A1, US 2004/0092907A1. The various parameters includingelectric field strength required for electroporation of any known celltype are generally known in the relevant research literature as well asnumerous patents and applications in the field. See e.g., U.S. Pat. Nos.6,678,556, 7,171,264, and 7,173,116. Apparatus for therapeuticapplication of electroporation are available commercially, e.g., theMedPulser™ DNA Electroporation Therapy System (Inovio/Genetronics, SanDiego, Calif.), and are described in patents such as U.S. Pat. Nos.6,567,694; 6,516,223, 5,993,434, 6,181,964, 6,241,701, and 6,233,482;electroporation may also be used for transfection of cells in vitro asdescribed e.g. in US20070128708A1. Electroporation may also be utilizedto deliver nucleic acids into cells in vitro. Accordingly,electroporation-mediated administration into cells of nucleic acidsincluding expression constructs utilizing any of the many availabledevices and electroporation systems known to those of skill in the artpresents an exciting new means for delivering an RNA of interest to atarget cell.

Sources of T Cells

In certain embodiments, a source of T cells is obtained from a subject.Non-limiting examples of subjects include humans, dogs, cats, mice,rats, and transgenic species thereof. Preferably, the subject is ahuman. T cells can be obtained from a number of sources, includingperipheral blood mononuclear cells, bone marrow, lymph node tissue,spleen tissue, umbilical cord, and tumors. In certain embodiments, anynumber of T cell lines available in the art, may be used. In certainembodiments, T cells can be obtained from a unit of blood collected froma subject using any number of techniques known to the skilled artisan,such as Ficoll separation. In one embodiment, cells from the circulatingblood of an individual are obtained by apheresis or leukapheresis. Theapheresis product typically contains lymphocytes, including T cells,monocytes, granulocytes, B cells, other nucleated white blood cells, redblood cells, and platelets. The cells collected by apheresis may bewashed to remove the plasma fraction and to place the cells in anappropriate buffer or media, such as phosphate buffered saline (PBS) orwash solution lacks calcium and may lack magnesium or may lack many ifnot all divalent cations, for subsequent processing steps. Afterwashing, the cells may be resuspended in a variety of biocompatiblebuffers, such as, for example, Ca-free, Mg-free PBS. Alternatively, theundesirable components of the apheresis sample may be removed and thecells directly resuspended in culture media.

In another embodiment, T cells are isolated from peripheral blood bylysing the red blood cells and depleting the monocytes, for example, bycentrifugation through a PERCOLL™ gradient. Alternatively, T cells canbe isolated from umbilical cord. In any event, a specific subpopulationof T cells can be further isolated by positive or negative selectiontechniques.

The cord blood mononuclear cells so isolated can be depleted of cellsexpressing certain antigens, including, but not limited to, CD34, CD8,CD14, CD19 and CD56. Depletion of these cells can be accomplished usingan isolated antibody, a biological sample comprising an antibody, suchas ascites, an antibody bound to a physical support, and a cell boundantibody.

Enrichment of a T cell population by negative selection can beaccomplished using a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. A preferred method iscell sorting and/or selection via negative magnetic immunoadherence orflow cytometry that uses a cocktail of monoclonal antibodies directed tocell surface markers present on the cells negatively selected. Forexample, to enrich for CD4+ cells by negative selection, a monoclonalantibody cocktail typically includes antibodies to CD14, CD20, CD11b,CD16, HLA-DR, and CD8.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain embodiments, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one embodiment, aconcentration of 2 billion cells/ml is used. In one embodiment, aconcentration of 1 billion cells/ml is used. In a further embodiment,greater than 100 million cells/ml is used. In a further embodiment, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet another embodiment, a concentration of cellsfrom 75, 80, 85, 90, 95, or 100 million cells/ml is used. In furtherembodiments, concentrations of 125 or 150 million cells/ml can be used.Using high concentrations can result in increased cell yield, cellactivation, and cell expansion.

T cells can also be frozen after the washing step, which does notrequire the monocyte-removal step. While not wishing to be bound bytheory, the freeze and subsequent thaw step provides a more uniformproduct by removing granulocytes and to some extent monocytes in thecell population. After the washing step that removes plasma andplatelets, the cells may be suspended in a freezing solution. While manyfreezing solutions and parameters are known in the art and will beuseful in this context, in a non-limiting example, one method involvesusing PBS containing 20% DMSO and 8% human serum albumin, or othersuitable cell freezing media. The cells are then frozen to −80° C. at arate of 1° per minute and stored in the vapor phase of a liquid nitrogenstorage tank. Other methods of controlled freezing may be used as wellas uncontrolled freezing immediately at −20° C. or in liquid nitrogen.

In one embodiment, the population of T cells is comprised within cellssuch as peripheral blood mononuclear cells, cord blood cells, a purifiedpopulation of T cells, and a T cell line. In another embodiment,peripheral blood mononuclear cells comprise the population of T cells.In yet another embodiment, purified T cells comprise the population of Tcells.

Expansion of T Cells

In certain embodiments, the T cells disclosed herein can be multipliedby about 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold,80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000 fold,4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, 10,000fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, or greater, and anyand all whole or partial integers therebetween. In one embodiment, the Tcells expand in the range of about 20 fold to about 50 fold.

Following culturing, the T cells can be incubated in cell medium in aculture apparatus for a period of time or until the cells reachconfluency or high cell density for optimal passage before passing thecells to another culture apparatus. The culturing apparatus can be ofany culture apparatus commonly used for culturing cells in vitro.Preferably, the level of confluence is 70% or greater before passing thecells to another culture apparatus. More preferably, the level ofconfluence is 90% or greater. A period of time can be any time suitablefor the culture of cells in vitro. The T cell medium may be replacedduring the culture of the T cells at any time. Preferably, the T cellmedium is replaced about every 2 to 3 days. The T cells are thenharvested from the culture apparatus whereupon the T cells can be usedimmediately or cryopreserved to be stored for use at a later time. Inone embodiment, the invention includes cryopreserving the expanded Tcells. The cryopreserved T cells are thawed prior to introducing nucleicacids into the T cell.

In another embodiment, the method comprises isolating T cells andexpanding the T cells. In another embodiment, the invention furthercomprises cryopreserving the T cells prior to expansion. In yet anotherembodiment, the cryopreserved T cells are thawed for electroporationwith the RNA encoding the chimeric membrane protein.

Another procedure for ex vivo expansion cells is described in U.S. Pat.No. 5,199,942 (incorporated herein by reference). Expansion, such asdescribed in U.S. Pat. No. 5,199,942 can be an alternative or inaddition to other methods of expansion described herein. Briefly, exvivo culture and expansion of T cells comprises the addition to thecellular growth factors, such as those described in U.S. Pat. No.5,199,942, or other factors, such as flt3-L, IL-1, IL-3 and c-kitligand. In one embodiment, expanding the T cells comprises culturing theT cells with a factor selected from the group consisting of flt3-L,IL-1, IL-3 and c-kit ligand.

The culturing step as described herein (contact with agents as describedherein or after electroporation) can be very short, for example lessthan 24 hours such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, or 23 hours. The culturing step as describedfurther herein (contact with agents as described herein) can be longer,for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.

Various terms are used to describe cells in culture. Cell culture refersgenerally to cells taken from a living organism and grown undercontrolled condition. A primary cell culture is a culture of cells,tissues or organs taken directly from an organism and before the firstsubculture. Cells are expanded in culture when they are placed in agrowth medium under conditions that facilitate cell growth and/ordivision, resulting in a larger population of the cells. When cells areexpanded in culture, the rate of cell proliferation is typicallymeasured by the amount of time required for the cells to double innumber, otherwise known as the doubling time.

Each round of subculturing is referred to as a passage. When cells aresubcultured, they are referred to as having been passaged. A specificpopulation of cells, or a cell line, is sometimes referred to orcharacterized by the number of times it has been passaged. For example,a cultured cell population that has been passaged ten times may bereferred to as a P10 culture. The primary culture, i.e., the firstculture following the isolation of cells from tissue, is designated P0.Following the first subculture, the cells are described as a secondaryculture (P1 or passage 1). After the second subculture, the cells becomea tertiary culture (P2 or passage 2), and so on. It will be understoodby those of skill in the art that there may be many population doublingsduring the period of passaging; therefore the number of populationdoublings of a culture is greater than the passage number. The expansionof cells (i.e., the number of population doublings) during the periodbetween passaging depends on many factors, including but is not limitedto the seeding density, substrate, medium, and time between passaging.

In one embodiment, the cells may be cultured for several hours (about 3hours) to about 14 days or any hourly integer value in between.Conditions appropriate for T cell culture include an appropriate media(e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15,(Lonza)) that may contain factors necessary for proliferation andviability, including serum (e.g., fetal bovine or human serum),interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM-CSF, IL-10,IL-12, IL-15, TGF-beta, and TNF-α, or any other additives for the growthof cells known to the skilled artisan. Other additives for the growth ofcells include, but are not limited to, surfactant, plasmanate, andreducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Mediacan include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, andX-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, andvitamins, either serum-free or supplemented with an appropriate amountof serum (or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells.Antibiotics, e.g., penicillin and streptomycin, are included only inexperimental cultures, not in cultures of cells that are to be infusedinto a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% CO₂).

The medium used to culture the T cells may include an agent that canco-stimulate the T cells. For example, an agent that can stimulate CD3is an antibody to CD3, and an agent that can stimulate CD28 is anantibody to CD28. This is because, as demonstrated by the data disclosedherein, a cell isolated by the methods disclosed herein can be expandedapproximately 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold,10,000 fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, or greater.In one embodiment, the T cells expand in the range of about 20 fold toabout 50 fold, or more by culturing the electroporated population.

In one embodiment, the method of expanding the T cells can furthercomprise isolating the expanded T cells for further applications. Inanother embodiment, the method of expanding can further comprise asubsequent electroporation of the expanded T cells followed byculturing. The subsequent electroporation may include introducing anucleic acid encoding an agent, such as a transducing the expanded Tcells, transfecting the expanded T cells, or electroporating theexpanded T cells with a nucleic acid, into the expanded population of Tcells, wherein the agent further stimulates the T cell. The agent maystimulate the T cells, such as by stimulating further expansion,effector function, or another T cell function.

Pharmaceutical Compositions

Pharmaceutical compositions of the present invention may comprise themodified cell or population of cells as described herein, in combinationwith one or more pharmaceutically or physiologically acceptablecarriers, diluents or excipients. Such compositions may comprise bufferssuch as neutral buffered saline, phosphate buffered saline and the like;carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol;proteins; polypeptides or amino acids such as glycine; antioxidants;chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminumhydroxide); and preservatives. Compositions of the present invention arepreferably formulated for intravenous administration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

The cells of the invention to be administered may be autologous,allogeneic or xenogeneic with respect to the subject undergoing therapy.

Cells of the invention can be administered in dosages and routes and attimes to be determined in appropriate pre-clinical and clinicalexperimentation and trials. Cell compositions may be administeredmultiple times at dosages within these ranges. Administration of thecells of the invention may be combined with other methods useful totreat the desired disease or condition as determined by those of skillin the art.

It can generally be stated that a pharmaceutical composition comprisingthe modified T cells described herein may be administered at a dosage of10⁴ to 10⁹ cells/kg body weight, in some instances 10⁵ to 10⁶ cells/kgbody weight, including all integer values within those ranges. T cellcompositions may also be administered multiple times at these dosages.The cells can be administered by using infusion techniques that arecommonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng.J. of Med. 319:1676, 1988). The optimal dosage and treatment regime fora particular patient can readily be determined by one skilled in the artof medicine by monitoring the patient for signs of disease and adjustingthe treatment accordingly.

The administration of the modified cells of the invention may be carriedout in any convenient manner known to those of skill in the art. Thecells of the present invention may be administered to a subject byaerosol inhalation, injection, ingestion, transfusion, implantation ortransplantation. The compositions described herein may be administeredto a patient transarterially, subcutaneously, intradermally,intratumorally, intranodally, intramedullary, intramuscularly, byintravenous (i.v.) injection, or intraperitoneally. In other instances,the cells of the invention are injected directly into a site ofinflammation in the subject, a local disease site in the subject, alymph node, an organ, a tumor, and the like.

It should be understood that the method and compositions that would beuseful in the present invention are not limited to the particularformulations set forth in the examples. The following examples are putforth so as to provide those of ordinary skill in the art with acomplete disclosure and description of how to make and use the cells,expansion and culture methods, and therapeutic methods of the invention,and are not intended to limit the scope of what the inventors regard astheir invention.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, fourth edition (Sambrook,2012); “Oligonucleotide Synthesis” (Gait, 1984); “Culture of AnimalCells” (Freshney, 2010); “Methods in Enzymology” “Handbook ofExperimental Immunology” (Weir, 1997); “Gene Transfer Vectors forMammalian Cells” (Miller and Calos, 1987); “Short Protocols in MolecularBiology” (Ausubel, 2002); “Polymerase Chain Reaction: Principles,Applications and Troubleshooting”, (Babar, 2011); “Current Protocols inImmunology” (Coligan, 2002). These techniques are applicable to theproduction of the polynucleotides and polypeptides of the invention,and, as such, may be considered in making and practicing the invention.Particularly useful techniques for particular embodiments will bediscussed in the sections that follow.

EXPERIMENTAL EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only, andthe invention is not limited to these Examples, but rather encompassesall variations that are evident as a result of the teachings providedherein.

The materials and methods employed in these experiments are nowdescribed.

gRNA DNA Target ID seq Position Strand Sequence PAM On CD2  #8 Ex3116760612 - ACAGCTGACAGGCTCGACAC TGG 63.4 (SEQ ID NO: 22) CD5  #4 Ex2- 61115090 + CGGCTCAGCTGGTATGACCC AGG 66.7 In2 (SEQ ID NO: 23) CD7 #85Ex2       670 - GGAGCAGGTGATGTTGACGG AGG 74.9 (SEQ ID NO: 24)

sgRNAs were designed to target CD2, CD5, or CD7 (e.g. SEQ ID NOs: 22-24,respectively) and synthesized using the GeneArt Precision sgRNAsynthesis kit. Cas9 expression plasmid (pGEM-Cas9) was amplified andlinearized. Cas9 RNA was synthesized using the mMessage mMachine T7Ultra kit. CRISPR editing was performed in Jurkat cells: CD2/CD5/CD7sgRNAs and Cas9 were transfected into Jurkat cells by electroporation.Expression of CD2/CD5/CD7 on Jurkat cells was detected by flow cytometryand the most effective CD2/CD5/CD7 sgRNAs were determined. CRISPRediting was then performed in primary human T cells using the mostefficient CD2/CD5/CD7 sgRNA: the chosen sgRNA and Cas9 RNA wereelectroporated into primary human T cells. CD2/CD5/CD7 expression wasdetected on the primary human T cells by flow cytometry to validate theknock-out/editing efficiency.

Specifically, fresh CD4/CD8 T cells were obtained and incubated withdynabeads on day 0. On day 4, cells were de-beaded then electroporatedwith Cas9 and sgRNA. Conditioned media (TCM (X-vivo15, human serum 5%,Glutamine), IL-7 10 ng/ml, and IL-15 10 ng/ml) was added to the cells.On day 6, cells were transduced with CAR lentivirus. On day 9, CARexpression was assessed. Cells were fed to 0.8e6/ml and frozen whenvolume <300 fl (FIG. 22 ).

CAR constructs: All constructs were generated using the lentiviral pTRPE4-1BB CD3zeta backbone. OKT11 CARs and TS2/18.1.1 CARs were constructedusing scFvs from antibodies generated from hybridomas purchased fromATCC (ATCC® CRL-8027™ and ATCC® HB-195™, respectively). The T11-2 CARwas constructed using an scFv from an antibody generated from ahybridoma that was received from Ellis Reinherz. All CD5 CARs wereconstructed using scFvs from antibody sequences published in WO2010/022737 A1, contents of which are incorporated by reference in theirentirety herein.

The results of the experiments are now described.

Example 1: A Novel Approach to Target T Cell Lymphomas and Leukemiaswithout Causing T Cell Toxicity

T-cell lymphomas and leukemias have an overall very poor prognosis, andthere are few therapeutic options available for these patients. Chimericantigen receptor T cell (CART) immunotherapy has led to unprecedentedresults in CD19+B-cell non-Hodgkin lymphoma (B-NHL). Herein, anothersuccessful “CART19-like” product was designed to target T-NHL. As CD19is not expressed in T-NHL, additional targets like CD2, CD5, CD7 andothers were evaluated for CART therapy. However, all these targets arealso expressed by normal T cells, leading to unacceptable clinicaltoxicity (T cell aplasia—immunodeficiency) (FIG. 1 ). Herein, a safe andeffective CART strategy was developed for the treatment of T-celllymphomas by editing the normal T cells to be resistant to CART killing(FIG. 2 ). CART therapy against T-cell lymphomas and leukemias wasfeasible when the CART target (CD2, CD5, CD7) was temporarily removedfrom normal T cells, thus avoiding CART-mediated killing andimmunodeficiency (FIG. 2 ).

Example 2: Anti-CD5 CAR T Cells (CART5) and CD5 Knocked-Out (KO) NormalT Cells

A two-pronged immunotherapy is disclosed herein that includes anti-CD5CAR T cells (CART5) and CD5 knocked-out (KO) normal T cells (FIG. 3 ).The CART5 destroys T cell lymphoma (e.g. T-NHL) or T cell leukemia cellsbut also kills normal T cells. The infusion of CD5 KO normal T cellsprovides CART-resistant T cell immunity until CART5 cells are depleted,in some cases by using a suicide gene (e.g. iCasp9, CD20/rituximab orothers).

CD5 was selected as a T-NHL target due to high expression on T-NHL cellsand absent expression in other tissues besides T cells and a minor Bcell subset. Six anti-CD5 CAR constructs were generated usingsingle-chain variable fragments (scFv) with different affinity (#17,#34, #9 with high, medium, low affinity respectively (Klitgaard J L, etal. (2013) British journal of haematology 163:182-93)) and expressed inT cells (FIGS. 4-5 ). Intriguingly, anti-CD5 CART did not require theknockout of CD5 to manufacture CART5 cells despite CD5 being expressedin 100% of CART cells (Mamonkin M, et. al. (2015) Blood 126:983-92),although CD5 expression was lower as compared to control T cells (FIG. 8). Without CRISPR-Cas9 KO of CD5, the CD5 mean fluorescence intensity(MFI) was 10 fold less in CART5 as compared to control T cells, whilethere was no change in another pan T-cell marker such as CD2 (FIG. 8 ).

In vitro and in vivo activity of the different CART5 constructs werecompared. Construct C3054, which was derived from the high-affinity scFv#17, demonstrated the best in vivo killing. Jurkat cells were transducedwith different CAR5 constructs (FIG. 13 , targeted epitope and affinityshown to the left) and with a GFP-NFAT reporter then co-cultured withCD5+ tumor cells (or controls) for 24 hours. The lead CART5 (C3054)shows increased NFAT activation (FIG. 13 ).

The lead anti-CD5 CART was implemented using a suicide system, and itsfunction was tested in vitro and in vivo. Without wishing to be bound byspecific theory, removal of CD5 (CRISPR-Cas9 KO) further increases CART5anti-tumor effect by eliminating the possible in cis surface interactionbetween CAR5 and CD5 on the CART5.

Insertion of a suicide system in the lead CART5 product: The leadcandidate CART5 (C3054) products are engineered to express a suicidesystem (FIG. 26 ). A P2A bicistronic vector encoding for both the CAR5and the inducible-caspase9 suicide system (Di Stasi A, et al. (2011)365:1673-83) (iCART5) is developed. Both orientations are cloned,CAR5-P2A-iCasp9 and iCasp9-P2A-CAR5 (FIG. 26 ), to define the mostefficient one (higher % double-expressing cells and lower % CAR5+iCasp9—for safety). iCART5 is efficiently eliminated using theclinical-grade compound rimiducid (AP1903, Bellicum Pharmaceuticals).

In vitro testing of iCART5: The newly generated iCART5 is compared to WTCART5 to confirm their efficacy (in vitro luciferase-based killing) andphenotype/function upon antigen stimulation (CD5+ Jurkat T-leukemiacells) (flow cytometry phenotype, 30-plex cytokine analysis by Luminexassay, CFSE proliferation and CD107a degranulation). Importantly, invitro depletion is tested by co-culturing iCART5 with differentconcentrations of rimiducid (0, 0.03, 0.3, 3, 10 nM) and checkingkilling at 15′, 30′ and at 2, 6, 12 and 24 hours. iCART5 is also testedagainst primary T-NHL cells using an established killing assay usingprimary CFSE-labelled Sezary cells.

In vivo testing of iCART5: In vivo xenograft models (Ruella M, et al.(2016) J Clin Invest) [using the click-beetle green (CBG)+T-leukemiacell line Jurkat cell line and click-beetle red (CBR)+ iCART5] are usedto test the ability of rimiducid (50 ug/mouse 23) to deplete iCART5 vs.WT CART5 in vivo. NOD SCID gamma-deficient mice (NSG) mice (8 per group)are injected with 2×10e⁶ CART5 cells/mouse. Tumor burden over time isassessed as bioluminescence (CBG) and T cell phenotype is studied byflow cytometry and expansion by bioluminescence (CBR) at multiple timepoints (hours/days). Mice are kept long-term (3-4 months) for survivaland monitoring of relapse. A human T-NHL xenograft model was previouslyestablised by injecting primary Sezary cells i.v. This model is used totest the iCART5 for both anti-tumor and depletion efficacies.

Evaluation of the role of CD5 KO in CART5: Preliminary data demonstratedthat CD5 CART5 are more effective than WT CART5 in vivo. CD5 is knockedout on CART5 cells using CRISPR-Cas9. The CRISPR-Cas9 CART expansionprotocol was previously optimized. Wild-type CART5 is compared to CD5 KOCART5 in vitro by testing CART5 viability, antigen-driven proliferation(using CFSE labeling), cytokine production (by 30-plex Luminex),degranulation (CD107a assessment by flow cytometry), cytotoxicity(luciferase-based), and phenotype (memory subsets, Th1/Th2). Both celllines (e.g., Jurkat) and primary samples are used as targets. In vivocomparison of CD5 KO vs. WT CART5 (1×10e6 cells/mouse) is performed inNSG mice bearing Jurkat and monitoring expansion and phenotype in theperipheral blood at day 10 and 14.

Mechanism of enhancement of CART5 function by CD5 KO: Having proven thatCD5KO improves CART5 activity, additional studies are performed tounderstand the mechanism: i. confocal imaging to analyze thelocalization of CAR5 and CD5 on CART5 cells; ii. single-molecule imaging(ONI nanoimager) to prove that CAR5 binds in cis to CD5; iii. expressingCAR5 in CD5+ Jurkat and show that CART5 fail to kill CAR5+ Jurkatbecause the CD5 epitope is masked (by CAR5); and iv. as CD5 as aninhibitory role on T cell activation, we will study CART5 activation inthe presence or not of CD5 (phospo-flow cytometry).

Generating CART-resistant normal T cells to avoid T cell aplasia: CART5are unable to distinguish neoplastic versus normal T cells as bothexpress similar levels of CD5. CART-resistant normal T cells aredeveloped to be co-infused with the anti-T-NHL CART to ensure immunityduring CART anti-tumor activity. CRISPR-Cas9 gene-editing is used toknock out CD5 in normal T cells thus making them invisible to CART5. Ahighly efficient CRISPR-Cas9 gRNA (#4) was generated that can KO ˜95% ofCD5 in normal T cells using an optimized CART expansion protocol. Datashow that CD5 KO T cells were resistant to CART5 while WT T cells (CD5+)were potently killed within 24 hours.

Manufacturing of CD5 KO normal T cells: CD5 KO normal T cells aredeveloped using a highly efficient gRNA (#4) that is electroporatedtogether with Cas9 protein (ThermoFisher v2) using a Lonza 4DNucleofector. CRISPR-Cas9 KO is performed on day 1, then cells arecultured at 30° C. for 2 days to increase gene-editing, then activatedwith anti-CD3/CD28 Dynabeads (beads::1 T cells) and expanded until theyreach a cell volume <300 fl.

In vitro evaluation of CD5 KO normal T cell resistance to iCART5killing: Resistance of CD5 KO normal T cells to CART5 is tested in vitroby performing killing assays (CFSE labeling of target T cells).Preliminary results showed that CD5 KO confers resistance. Threeadditional T cell donors will be tested.

In vivo evaluation of CD5 KO normal T cell resistance to iCART5 in anautologous xenograft model: NSG mice (8 mice/group) are engrafted withluciferase+CD5 KO normal T cells or WT, and after two days autologousiCART5 is injected. The effect of iCART5 on CD5 ko and WT normal T cellsis assessed by bioluminescence. Once WT T cells are completelyeliminated by iCART5 (luminescence) rimiducid is administered to depleteiCART5. Then WT T cells are reinjected to demonstrate that normal Tcells can repopulate the host. Mice are bled weekly to assess CARTexpansion.

Evaluation of the role of CD5 KO on normal T cell functions: The role ofCD5 KO is investigated in normal T cells by carefully studying T celleffector functions. After TCR-specific stimulation (anti-CD3/CD28 beads)cytokine production (30-plex Luminex), proliferation (CF SE) andactivation of CD5KO T cells vs. WT are measured. Also tested are whetherCD5KO T cells proliferate and produce similarly to WT when exposed tocommon infections.

Defining the optimal CD5KO normal T cell dose for clinical use: Usingboth in silico and experimental approaches (TCR sequencing and tetramerstaining of TCRs specific for infectious agents) the minimum number ofcells to be infused in relapsed or refractor (r/r) T-NHL patients isdefined in order to ensure sufficient T cell immunity to the most commoninfections.

Testing the concurrent infusion of iCART5 and CD5 KO normal T cells in aphase 1 pilot clinical trial for patients with advanced T cell lymphoma:An Investigation New Drug (IND) package is developed and submitted tothe FDA. A phase 1 clinical trial is started to test the anti-T-NHL CARTapproach in patients. The IND package is based on preliminary resultsand further data from experiments described herein.

Clinical trial protocol design: The phase 1 clinical trial includespatients with r/r T-NHL that are treated using a 3+3 protocol design.From a single apheresis, two products are generated using enriched Tcells: #1. CRISPR-Cas9 CD5 KO normal T cells and #2. iCART5. The firstproduct to be infused is the KO normal T cells followed the next day byiCART5. The first cohort of patients receives lymphodepletion[cyclophosphamide (60 mg/kg×2 days) and fludarabine (25 mg/m2×5 days)]and product #1. If no dose-limiting toxicity (DLT) is observed, Cohort 2receives lymphodepletion, product #1 and 1-5×10e⁷ total iCART5 (product#2) over 3 days (10%, 30%, 60% of the total dose); 1-5×10e⁷ total CART5is a suboptimal dose based on CART19 experiments in B-NHL8. If no DLT isobserved in cohort 2, cohort 3 receives lymphodepletion, product #1 and1-5×10e⁸ total CART5 (full dose). Patient from cohort #1 will is allowedto proceed to cohort #2 if no toxicity is observed within the first 4weeks. Based on tumor clearance (and maximum at month 6) the iCART5cells are depleted using the dimerizing agent rimiducid (NCT02744287) toprevent possible long-term T cell toxicity.

Preparation of IND package and FDA submission: Optimization ofclinical-grade manufacturing is run in collaboration with the ClinicalVaccine and Cell Production Facility (CVPF). The results of alldescribed preclinical experiments together with the clinical trialprotocol are formatted to fit an IND application. Extensive support forthe IND preparation is available within the CCI and ACC.

Patient enrollment and treatment: After successful submission of the INDand approval from all the regulatory agencies, the phase 1 trial isstarted at the University of Pennsylvania, within the Lymphoma Program(Director: Dr. Stephen Schuster; Scientific Director: Dr. Marco Ruella).The Lymphoma program has a dedicated clinical research unit (CRU) withextensive experience managing early-stage studies. Dr. Ruella is thePrincipal Investigator of this trial with Dr. Carl June being theScientific Protocol Advisor. Manufacturing of the 2 products isperformed at the CVPF.

Correlative studies: Patient samples (peripheral blood) are analyzed atmultiple time points (apheresis, day −1, 0, 7, 14, 28, 60, 90) to testCART expansion (qPCR and flow cytometry), CART phenotype (CyTOF), CARTgene expression profiling (GEP) (NanoString, single-cell RNAseq 10×Genomics) and cytokine levels in the serum (Luminex, 30-plex array).Additional studies are performed on tumor biopsies pre-treatment andpost-treatment when available (RNAseq and Hyperion analysis of the tumormicroenvironment).

This trial will be a key milestone in the development of novel combinedimmunotherapies as it represents an innovative immunotherapeuticapproach to treat T cell non-Hodgkin lymphomas avoiding toxicity.Anti-CD5 CAR T cells kill tumor T cells but unavoidably also normal Tcells due to similar CD5 expression. However, the strategy describedherein includes the co-infusion of normal T cells that have been knockedout for CD5, thereby ensuring T cell immunological protection duringCART5 anti-tumor activity. CART5 cells are then depleted using a suicidesystem to ensure normal immunological reconstitution long term. This isone of the first CART trials for T-NHL and the only one including atwo-pronged approach addressing the issue of toxicity. T-NHL have a verypoor prognosis, and there are currently no active immunotherapiesavailable. Therefore the development of such an innovative strategyrepresents a vertical advance in the field of hematology andimmunotherapy. Based on the clinical results of the phase 1 trials andthe findings of the correlative studies this strategy can also beimplemented to targeting multiple targets at the same time to avoidantigen-loss escape (e.g., CART5+ CART7) or combine CART with smallmolecules that can enhance CART-mediated killing.

Example 3: Anti-CD2 CAR T Cells (CART2) and CD2 Knocked-Out (KO) NormalT Cells

A two-pronged immunotherapy approach is disclosed herein that includesanti-CD2 CAR T cells (CART2) and CD2 knocked-out (KO) normal T cells(FIG. 3 ). The CART2 destroys T cell lymphoma (e.g. T-NHL) or T cellleukemia cells, but also kills normal T cells. Infusion of CD2 KO normalT cells provides CART-resistant T cell immunity until CART2 cells aredepleted, in some cases by using a suicide gene (e.g. iCasp9).

Guide RNAs were designed to knock out the CD2 gene (and CD5) using theCRISPR/Cas9 system. CD2 was effectively knocked out 78% of the T cellpopulation. Second generation anti-CD2 and anti-CD5 CARs (CART2 andCART5, respectively) were generated (FIG. 4 ). Knock out cells (CD2KOand CD5KO) were incubated with their corresponding CART cells (CART2 andCART5, respectively), stimulated, and population doublings measured(FIG. 7 ). Mock electroporated cells that did not contain gRNA were usedas a control for comparison. Without CD2 KO CART2 cells would not expand(FIG. 7 ). With KO CART2 and CART5 reach about 5-8 population doublings(FIG. 7 ).

Jurkat cells were transduced with the different CAR2 constructs and witha GFP-NFAT reporter then co-cultured with CD2+ tumor cells (or controls)for 24 hours. The lead CART2 (C3043) shows increased NFAT activation(FIG. 14 ).

The CART expansion protocol was optimized CART2 and CART5 cellscontinued to expand up to 18 days when incubated with CD2KO or CD5 KOcells (FIG. 9 ).

Example 4: CART2 and CART5 Testing

FIG. 6 illustrates the CD5 (or CD2, or CD7) KO manufacturing process andCRISPR-Cas9 KO efficiency.

The 6 different CAR2 and 6 CAR5 constructs were challenged in vitro byco-culturing them with luciferase+ Jurkat cells (T-cell leukemia cellline). At 24 hrs, total killing was measured as relative reduction inluminescence. For CART2 only #3029, #3030 and #3043 show anti-tumoreffect. (FIG. 10 ). For CART5, all the constructs demonstratedanti-tumor effects (FIG. 11 ). Lead CART candidates (CART2 C3043 andCART5 C3054) were selected and tested. The effects of CD2 or CD5knock-out on CART function was tested). CART2- and CART5-resistant Tcells were successfully generated (CD5 and CD2 were knocked-out innormal T cells.

CART2 and CART5 activity against cutaneous T cell lymphoma was tested.Twenty-four hour killing assays were performed. CART2 cells were activeagainst primary Sezary cells (leukemic Cutaneous T Cell Lymphoma) andthe HH Sezary cell line (FIG. 15 ). Also CART5 were active against HHcells (FIG. 15 ).

In vivo efficacy of CART2 and CART5 was measured. NSG mice wereengrafted with Luciferase+ Jurkat cells and mice were randomized toreceive control T cells or CART2 or CART5 (1×10⁶) at day 7. Mice wereimaged weekly using the IVIS Xenogen Spectrum and analyzed withLivingImage software. CART2 C3043 and CART5 C3054 were the mosteffective (FIG. 12 ).

CART2 and CART5 were demonstrated to recognize normal T cells(autologous and allogeneic) and kill them (FIG. 16 ).

It was also demonstrated that removal of the CAR target protects normalT cells from CART killing (FIG. 17 ). CD5 KO but not WT normal T cellswere resistant to CART5 killing. Normal resting T cells are recognizedand killed by CART2 (FIG. 17 , top) and CART5 (FIG. 17 , bottom).Efficient KO of CD2 or CD5 from normal T cells using CRISPR-Cas9 led toresistance to CART2 or CART5 killing respectively (FIG. 17 ).

CMV-specific T cells were present in CD2KO and CD5KO normal T cellproducts (FIG. 18 ). CD2 and CD5 KO normal T cells maintained theability to recognize CMV peptides and produce cytokines (FIG. 18 ;HLA-A-02:01-CMV PP65 NLVPMVATV dextramer (SEQ ID NO: 101); ICS after 4 hexposure to CETF peptides. After secondary culture with CMV-peptidepulsed APC).

Example 5: Anti-CD7 CAR T Cells (CART7) and CD7 Knocked-Out (KO) NormalT Cells

A two-pronged immunotherapy approach is disclosed herein that includesanti-CD7 CAR T cells (CART7) and CD7 knocked-out (KO) normal T cells.The CART7 destroys T cell lymphoma (e.g. T-NHL) or T cell leukemiacells, but also kills normal T cells. Infusion of CD7 KO normal T cellsprovides CART-resistant T cell immunity until CART7 cells are depleted,in some cases by using a suicide gene (e.g. iCasp9).

Guide RNAs were designed to knock out the CD7 gene using the CRISPR/Cas9system. CD7 was effectively knocked out 79% of the T cell population(FIG. 25 ). Six anti-CD7 CAR5 were generated (FIG. 25 ).

Example 6: Dual Specific CAR T Cells

Two lentiviral constructs were generated that included CAR5 (C3054) andCAR2 (C3043) linked by a P2A sequence (FIG. 19 ). Gene expression wasdriven by an EFlalpha promoter. The CAR5 construct has 4-1BBcostimulatory and CD3zeta signaling domains. Efficient knock-out of bothCD2 and CD5 in normal T cells was demonstrated (FIGS. 20A-20B).

Example 6: CD5 KO Enhances CART Immunotherapy

It was demonstrated that CD5 KO CART5s are more effective than CD5+CART5s in vivo. CD5 KO increased CART5 anti-tumor efficacy (FIG. 21 ).In a Jurkat T-ALL xenograft model using NSG mice, CD5 KO CART5 (2×10⁶cells/mouse) lead to complete long-term responses and longer survival ascompared to WT CART5 (FIG. 21 ).

CD5 KO CART19 were also more effective than CD5+ CART19 in vivo. CD5 KOincreased CART19 anti-tumor efficacy (FIG. 22 ). In a NALM6 B-ALLxenograft model CD5 KO CART19 exhibited drastically higher tumor controlas compared to WT CART19 (FIG. 22 ).

CART5 and CART2 were also capable of targeting 20% of AML. CART2 cellswere co-cultured with CD2+ AML cells and showed significant killing at24 hours (FIGS. 23A-23B).

CART5 also targeted 100% of CLL and MCL. A cytotoxicity assay wasperformed and demonstrated that CART5 cells can recognize and kill CD5+MCL cell lines (Jeko-1 and Mino) (FIG. 24 ).

These data demonstrate that knocking out CD5 enhances CART therapy whentreating with an anti-CD5 CAR, or surprisingly when treating with adifferent CAR T cell (e.g. a CD19 CART cell).

OTHER EMBODIMENTS

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

What is claimed is:
 1. A pharmaceutical composition comprising a firstpopulation of T-cells comprising a chimeric antigen receptor (CAR) and amutated endogenous CD5 gene and a second population of T-cellscomprising a mutated endogenous CD5 gene, wherein the second populationdoes not comprise the CAR.
 2. The pharmaceutical composition of claim 1,wherein the mutated endogenous CD5 gene is a gene edited endogenous CD5gene.
 3. The pharmaceutical composition of claim 1, wherein the firstand second population of T-cells comprising the mutated endogenous CD5gene has a decrease in expression of endogenous CD5 protein.
 4. Thepharmaceutical composition of claim 1, wherein the chimeric antigenreceptor comprises an antigen binding domain that binds to CD5, CD19,CD2, CD7, a tumor-specific antigen (TSA), a tumor associated antigen(TAA), a glioma-associated antigen, carcinoembryonic antigen (CEA),b-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactiveAFP, thyroglobulin, RAGE-I, MN-CA IX, human telomerase reversetranscriptase, RUI, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2,M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1,LAGE-1a, p53, prostein, PSMA, Her2/neu, survivin, telomerase,prostate-carcinoma tumor antigen-I (PCTA-1), MAGE, ELF2M, neutrophilelastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-Ireceptor, mesothelin, MART-I/Mel an A (MART-1), gplOO (Pmel 17),tyrosinase, TRP-1, TRP-2, MAGE-I, MAGE-3, BAGE, GAGE-I, GAGE-2, p15,Ras, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, EBVA, HPV antigen E6,HPV antigen E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO,p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4,CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F,5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029,FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K,NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilinC-associated protein, TAAL6, TAG72, TLP, or TPS.
 5. The pharmaceuticalcomposition of claim 1, wherein the chimeric antigen receptor comprisesan antigen binding domain that binds to CD5.
 6. The pharmaceuticalcomposition of claim 5, wherein the antigen binding domain of the CARcomprises a complementarity determining region (CDR) comprising theamino acid sequence selected from the group consisting of SEQ ID NOs:31-36, 43-48, 53-58, 65-70, 83-88, and 95-100.
 7. The pharmaceuticalcomposition of claim 5, wherein the antigen binding domain of the CARcomprises an scFv comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 27, 28, 39, 40, 50, 61, 62, 73, 74, 79,80, 91, and
 92. 8. The pharmaceutical composition of claim 1, whereinthe first population of T-cells comprises more than one chimeric antigenreceptor.
 9. The pharmaceutical composition of claim 1, wherein themutated CD5 gene is a CRISPR mutated CD5 gene.
 10. The pharmaceuticalcomposition of claim 1, wherein the CAR comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 25, 26, 37, 38, 49,59, 60, 71, 72, 77, 78, 89, and
 90. 11. A method of treating cancer in asubject, the method comprising administering to the subject thepharmaceutical composition of claim
 1. 12. The method of claim 11,wherein the cancer is a T cell lymphoma or a T cell leukemia.
 13. Themethod of claim 11, wherein the cancer is acute myeloid leukemia (AML),T-cell acute lymphoblastic leukemia (T-ALL), acute lymphoblasticleukemia (ALL), or chronic lymphocytic leukemia (CLL).
 14. Thepharmaceutical composition of claim 1, wherein the chimeric antigenreceptor does not comprise an antigen binding domain that binds to CD5.15. A method of enhancing the efficacy of a T-cell comprising a chimericantigen receptor for treating cancer in a subject, the method comprisingadministering to the subject a T-cell comprising a mutated endogenousCD5 gene and the chimeric antigen receptor, wherein the chimeric antigenreceptor does not comprise an antigen binding domain that binds to CD5.16. The method of claim 15, wherein the mutated endogenous CD5 gene is aknock-out of the endogenous CD5 gene.
 17. The method of claim 15,wherein the chimeric antigen receptor comprises an antigen bindingdomain that binds to CD5, CD19, CD2, CD7, a tumor-specific antigen(TSA), a tumor associated antigen (TAA), a glioma-associated antigen,carcinoembryonic antigen (CEA), b-human chorionic gonadotropin,alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1,MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS),intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase,prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein,PSMA, Her2/neu, survivin, telomerase, prostate-carcinoma tumor antigen-1(PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulingrowth factor (IGF)-I, IGF-II, IGF-I receptor, mesothelin, MART-1/Mel anA (MART-1), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3,BAGE, GAGE-1, GAGE-2, p15, Ras, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK,MYL-RAR, EBVA, HPV antigen E6, HPV antigen E7, TSP-180, MAGE-4, MAGE-5,MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA,TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4,Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG,BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50,CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50,MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 bindingprotein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, or TPS. 18.A chimeric antigen receptor (CAR), wherein the CAR comprises an antigenbinding domain that binds to CD5, a transmembrane domain, and anintracellular domain, wherein the antigen binding domain comprises acomplementarity determining region (CDR) comprising the amino acidsequence selected from the group consisting of SEQ ID NOs: 83-88 and95-100.
 19. The chimeric antigen receptor of claim 18, wherein the CARcomprises an scFv comprising the amino acid sequence selected from thegroup consisting of SEQ ID NOs: 73, 74, 79, 80, 91, and
 92. 20. Thechimeric antigen receptor of claim 18, wherein the CAR comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:71, 72, 77, 78, 89, and 90.